Deactivation of the lipopolysaccharide antagonist eritoran (E5564) by high-density lipoprotein-associated apolipoproteins

Lipid A, the active moiety of LPS, exerts its effects through interaction with TLR4, triggering a signalling cascade that results in the release of pro-inflammatory cytokines. Eritoran is a lipid A analogue that competes with LPS for binding to TLR4; however, after intravenous administration, it undergoes a time-dependent deactivation as a consequence of binding to high-density lipoproteins (HDLs). The site of eritoran association with HDL remains unknown. Therefore the aim of this study was to determine if HDL-associated apolipoproteins A1, A2, serum amyloid A (SAA) and C1, inhibit the ability of eritoran to block LPS-induced TNF-α release from whole blood. Eritoran activity after LPS stimulation in human whole blood was assessed in the presence of reconstituted HDL (rHDL) containing different apos. In rHDL, the major apolipoproteins in both the healthy and septic state, A1 and SAA, caused a significant reduction in eritoran antagonistic activity and had a greater effect than minor apolipoproteins A2 and C1. Apolipoproteins associated with HDL are likely to facilitate eritoran deactivation. Apolipoproteins A1 and SAA should be of particular focus as they are the major apos found on HDL in both the healthy and septic state. Further evaluation of the physical association between apolipoproteins and eritoran should be explored.     

Atlantic snake pipefish (<Emphasis Type="Italic">Entelurus aequoreus</Emphasis>) extends its northward distribution range to Svalbard (Arctic Ocean)

Ecological forecasts predict the immigration of boreal species into Arctic waters as one consequence of rising sea temperatures. Here, we report the finding of Atlantic snake pipefish (Entelurus aequoreus) off the western coast of Spitsbergen at 79°N in August 2006. This syngnathid fish species, which was presumed to be confined to waters south of Iceland, has dramatically increased in population size in its core distribution area in the northeastern Atlantic since 2002, probably in response to greater reproduction success due to higher water temperatures. We conclude that our finding is an indication of the predicted northward extension of the distribution range of boreal species.     

Phosphorylation modulates the function of the calcium release channel of sarcoplasmic reticulum from skeletal muscle.

The modulation of the calcium release channel (CRC) by protein kinases and phosphatases was studied. For this purpose, we have developed a microsyringe applicator to achieve sequential and multiple treatments with highly purified kinases and phosphatases applied directly at the bilayer surface. Terminal cisternae vesicles of sarcoplasmic reticulum from rabbit fast twitch skeletal muscle were fused to planar lipid bilayers, and single-channel currents were measured at zero holding potential, at 0.15 microM free Ca2+, +/- 0.5 mM ATP and +/- 2.6 mM free Mg2+. Sequential dephosphorylation and rephosphorylation rendered the CRC sensitive and insensitive to block by Mg2+, respectively. Channel recovery from Mg2+ block was obtained by exogenous protein kinase A (PKA) or by Ca2+/calmodulin-dependent protein kinase II (CalPK II). Somewhat different characteristics were observed with the two kinases, suggesting two different states of phosphorylation. Channel block by Mg2+ was restored by dephosphorylation using protein phosphatase 1 (PPT1). Before application of protein kinases or phosphatases, channels were found to be "dephosphorylated" (inactive) in 60% and "phosphorylated" (active) in 40% of 51 single-channel experiments based on the criterion of sensitivity to block by Mg2+. Thus, these two states were interconvertable by treatment with exogenously added protein kinases and phosphatases. Endogenous Ca2+/calmodulin-dependent protein kinase (end CalPK) had an opposite action to exogenous CalPK II. Previously, dephosphorylated channels using PPT (Mg2+ absent) were blocked in the closed state by action of endogenous CalPK. This block was removed to normal activity by the action of either PPT or by exogenous CalPK II. Our findings are consistent with a physiological role for phosphorylation/dephosphorylation in the modulation of the calcium release channel of sarcoplasmic reticulum from skeletal muscle. A corollary of our studies is that only the phosphorylated channel is active under physiological conditions (mM Mg2+). Our studies suggest that phosphorylation can be at more than one site and, depending on the site, can have different functional consequences on the CRC.     

No reduction in genetic diversity of Swiss stone pine (Pinus cembra L.) in Tatra Mountains despite high fragmentation and small population size

In Europe, most of the alpine timberline ecotone has been altered by human activities and climate change. Hence, mountain forests are of the highest conservation interest. Here, we screened 25 populations of Swiss stone pine (Pinus cembra L.) from the Carpathians and the Alps, using a set of ten microsatellite primers to assess the relative conservation value of populations sampled in Polish and Slovak Tatra National Parks, where potential extinction risk is the highest within the Carpathian range. Although endangered, with small and fragmented populations, P. cembra in the Tatra Mts. shows high levels of allelic richness (AR = 5.0) and observed heterozygosity (H _o  = 0.554). Our results suggest that anthropogenic habitat fragmentation has had little impact on DNA variation of Swiss stone pine in the Tatra Mts. However, the effects of changing conditions on the genetic structure may occur with a substantial time delay due to the long life span of P. cembra. Moreover, inbreeding depression may occur in the next generations, since we found inbreeding (F _IS  = 0.063) and elevated coancestry coefficient (θ = 0.062) in all populations. Also a shallow pattern of genetic differentiation between populations was found, indicating recent fragmentation of a common gene pool that formerly occupied a larger range. Therefore, the Tatra Mts. can be considered as a single conservation unit. Based on our results, we suggest possible conservation activities for Swiss stone pine both in Poland and Slovakia.     

The ecological-evolutionary interplay: density-dependent sexual selection in a migratory songbird

Little is understood about how environmental heterogeneity influences the spatial dynamics of sexual selection. Within human-dominated systems, habitat modification creates environmental heterogeneity that could influence the adaptive value of individual phenotypes. Here, we used the gray catbird to examine if the ecological conditions experienced in the suburban matrix (SM) and embedded suburban parks (SP) influence reproductive strategies and the strength of sexual selection. Our results show that these habitats varied in a key ecological factor, breeding density. Moreover, this ecological factor was closely tied to reproductive strategies such that local breeding density predicted the probability that a nest would contain extra-pair offspring. Partitioning reproductive variance showed that while within-pair success was more important in both habitats, extra-pair success increased the opportunity for sexual selection by 39% at higher breeding densities. Body size was a strong predictor of relative reproductive success and was under directional selection in both habitats. Importantly, our results show that the strength of sexual selection did not differ among habitats at the landscape scale but rather that fine-scale variation in an ecological factor, breeding density, influenced sexual selection on male phenotypes. Here, we document density-dependent sexual selection in a migratory bird and hypothesize that coarse-scale environmental heterogeneity, in this case generated by anthropogenic habitat modification, changed the fine-scale ecological conditions that drove the spatial dynamics of sexual selection.     

Preliminary analysis of mitochondrial DNA variation in a southern feeding group of eastern North Pacific gray whales

Although the majority of eastern North Pacific(ENP) gray whales migrate to feeding grounds inthe Bering and Chukchi Seas, some terminate themigration in more southerly areas such asBritish Columbia (BC). Long-term sightingstudies in Clayoquot Sound (CS), BC, indicatethat approximately 35–50 individuals exhibitlong-term fidelity to this site. To determinethe sex composition (based on genetic sexing)of CS gray whales and to assess whethermatrilineal site-fidelity occurs in CS, wecollected skin biopsy samples from 16 CSindividuals (`residents') and 41 samples fromother areas (representative of the overallpopulation in the ENP: `non-residents'). Atotal of 27 polymorphic sites defined 24haploytpes among the 57 samples sequenced forHV1 of the mtDNA control region. Thenucleotide and haplotype diversities of thesesamples were 0.017 (SE = 0.0012) and 0.94(SE = 0.0019), respectively. Neighbor-joininganalysis revealed five lineages each of whichcontained haplotypes that were observed in bothresidents and non-residents. Residents did notdiffer significantly from non-residents, and nosignificant sex-ratio bias was found. Thesedata suggest a level of diversity that isinconsistent with a severe historicalbottleneck, and given the available samplesize, do not indicate matrilineally directedfidelity to Clayoquot Sound.     

Rapid, independent evolution of flightlessness in four species of Pacific Island rails (Rallidae): an analysis based on mitochondrial sequence data

Flightless rails were once ubiquitous in the avifauna of Pacific oceanic islands. Most species have become extinct since human colonization of islands began about 2000 years ago. In this study, we use mitochondrial sequence data to estimate the phylogenetic relationships and ages of four species of flightless insular rails in the genus Porzana : palmeri , from Laysan Island in the Hawaiian archipelago; sandwichensis , from the island of Hawaii; monasa , from Kosrae Island in Micronesia; and atra , from Henderson Island in the Pitcairn group. Although all four species survived into historic times, all but atra are now extinct. The optimal trees show that palmeri is descended from Porzana pusilla , a volant crake distributed widely throughout the Old World. Porzana sandwichensis , P. monasa , and P. atra are each descended from the lineage leading to P. tabuensis , a volant rail widespread in northern and eastern Australia and on islands north to Micronesia and the Philippines and east through Polynesia. Loss of flight appears to have evolved rapidly in these insular rails, based on both sequence divergence values and data on the ages of the islands. In the case of the Laysan Rail ( palmeri ), divergences including loss of flight probably evolved in less than 125,000 years .     

Multimodal pressure-flow method to assess dynamics of cerebral autoregulation in stroke and hypertension

Background

This study evaluated the effects of stroke on regulation of cerebral blood flow in response to fluctuations in systemic blood pressure (BP). The autoregulatory dynamics are difficult to assess because of the nonstationarity and nonlinearity of the component signals.

Methods

We studied 15 normotensive, 20 hypertensive and 15 minor stroke subjects (48.0 ± 1.3 years). BP and blood flow velocities (BFV) from middle cerebral arteries (MCA) were measured during the Valsalva maneuver (VM) using transcranial Doppler ultrasound.

Results

A new technique, multimodal pressure-flow analysis (MMPF), was implemented to analyze these short, nonstationary signals. MMPF analysis decomposes complex BP and BFV signals into multiple empirical modes, representing their instantaneous frequency-amplitude modulation. The empirical mode corresponding to the VM BP profile was used to construct the continuous phase diagram and to identify the minimum and maximum values from the residual BP (BP_R) and BFV (BFV_R) signals. The BP-BFV phase shift was calculated as the difference between the phase corresponding to the BP_R and BFV_R minimum (maximum) values. BP-BFV phase shifts were significantly different between groups. In the normotensive group, the BFV_R minimum and maximum preceded the BP_R minimum and maximum, respectively, leading to large positive values of BP-BFV shifts.

Conclusion

In the stroke and hypertensive groups, the resulting BP-BFV phase shift was significantly smaller compared to the normotensive group. A standard autoregulation index did not differentiate the groups. The MMPF method enables evaluation of autoregulatory dynamics based on instantaneous BP-BFV phase analysis. Regulation of BP-BFV dynamics is altered with hypertension and after stroke, rendering blood flow dependent on blood pressure.

     

Coenzyme binding by 3-hydroxybutyrate dehydrogenase, a lipid-requiring enzyme: lecithin acts as an allosteric modulator to enhance the affinity for coenzyme

The role of phospholipid in the binding of coenzyme, NAD(H), to 3-hydroxybutyrate dehydrogenase, a lipid-requiring membrane enzyme, has been studied with the ultrafiltration binding method, which we optimized to quantitate weak ligand binding (KD in the range 10-100 microM). 3-Hydroxybutyrate dehydrogenase has a specific requirement of phosphatidylcholine (PC) for optimal function and is a tetramer quantitated both for the apodehydrogenase, which is devoid of phospholipid, and for the enzyme reconstituted into phospholipid vesicles in either the presence or absence of PC. We find that (i) the stoichiometry for NADH and NAD binding is 0.5 mol/mol of enzyme monomer (2 mol/mol of tetramer); (ii) the dissociation constant for NADH binding is essentially the same for the enzyme reconstituted into the mixture of mitochondrial phospholipids (MPL) (KD = 15 +/- 3 microM) or into dioleoyl-PC (KD = 12 +/- 3 microM); (iii) the binding of NAD+ to the enzyme-MPL complex is more than an order of magnitude weaker than NADH binding (KD approximately 200 microM versus 15 microM) but can be enhanced by formation of a ternary complex with either 2-methylmalonate (apparent KD = 1.1 +/- 0.2 microM) or sulfite to form the NAD-SO3- adduct (KD = 0.5 +/- 0.1 microM); (iv) the binding stoichiometry for NADH is the same (0.5 mol/mol) for binary (NADH alone) and ternary complexes (NADH plus monomethyl malonate); (v) binding of NAD+ and NADH together totals 0.5 mol of NAD(H)/mol of enzyme monomer, i.e., two nucleotide binding sites per enzyme tetramer; and (vi) the binding of nucleotide to the enzyme reconstituted with phospholipid devoid of PC is weak, being detected only for the NAD+ plus 2-methylmalonate ternary complex (apparent KD approximately 50 microM or approximately 50-fold weaker binding than that for the same complex in the presence of PC). The binding of NADH by equilibrium dialysis or of spin-labeled analogues of NAD+ by EPR spectroscopy gave complementary results, indicating that the ultrafiltration studies approximated equilibrium conditions. In addition to specific binding of NAD(H) to 3-hydroxybutyrate dehydrogenase, we find significant binding of NAD(H) to phospholipid vesicles. An important new finding is that the nucleotide binding site is present in 3-hydroxybutyrate dehydrogenase in the absence of activating phospholipid since (a) NAD+, as the ternary complex with 2-methylmalonate, binds to the enzyme reconstituted with phospholipid devoid of PC and (b) the apodehydrogenase, devoid of phospholipid, binds NADH or NAD-SO3- weakly (half-maximal binding at approximately 75 microM NAD-SO3- and somewhat weaker binding for NADH).(ABSTRACT TRUNCATED AT 400 WORDS)