New hypotheses about the structure–function of proprotein convertase subtilisin/kexin type 9: Analysis of the epidermal growth factor‐like repeat A docking site using WaterMap

LDL cholesterol (LDL‐C) is cleared from plasma via cellular uptake and internalization processes that are largely mediated by the low‐density lipoprotein cholesterol receptor (LDL‐R). LDL‐R is targeted for lysosomal degradation by association with proprotein convertase subtilisin‐kexin type 9 (PCSK9). Gain of function mutations in PCSK9 can result in excessive loss of receptors and dyslipidemia. On the other hand, receptor‐sparing phenomena, including loss‐of‐function mutations or inhibition of PCSK9, can lead to enhanced clearance of plasma lipids. We hypothesize that desolvation and resolvation processes, in many cases, constitute rate‐determining steps for protein–ligand association and dissociation, respectively. To test this hypothesis, we analyzed and compared the predicted desolvation properties of wild‐type versus gain‐of‐function mutant Asp374Tyr PCSK9 using WaterMap, a new in silico method for predicting the preferred locations and thermodynamic properties of water solvating proteins (“hydration sites”). We compared these results with binding kinetics data for PCSK9, full‐length LDL‐R ectodomain, and isolated EGF‐A repeat. We propose that the fast k_on and entropically driven thermodynamics observed for PCSK9‐EGF‐A binding stem from the functional replacement of water occupying stable PCSK9 hydration sites (i.e., exchange of PCSK9 H‐bonds from water to polar EGF‐A groups). We further propose that the relatively fast k_off observed for EGF‐A unbinding stems from the limited displacement of solvent occupying unstable hydration sites. Conversely, the slower k_off observed for EGF‐A and LDL‐R unbinding from Asp374Tyr PCSK9 stems from the destabilizing effects of this mutation on PCSK9 hydration sites, with a concomitant increase in the persistence of the bound complex. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Interactions between adenosine and K+ channel-related pathways in the coupling of somatosensory activation and pial arteriolar dilation

Multiple, perhaps interactive, mechanisms participate in the linkage between increased neural activity and cerebral vasodilation. In the present study, we assessed whether neural activation-related pial arteriolar dilation (PAD) involved interactions among adenosine (Ado) A(2) receptors (A(2)Rs), large-conductance Ca(2+)-operated K(+) (BK(Ca)) channels, and inward rectifier K(+) (K(ir)) channels. In rats with closed cranial windows, we monitored sciatic nerve stimulation (SNS)-induced PAD in the absence or presence of pharmacological blockade of A(2)Rs (ZM-241385), ecto-5'-nucleotidase (α,β-methylene-adenosine diphosphate), BK(Ca) channels (paxilline), and K(ir) channels (BaCl(2)). Individually, these interventions led to 53-66% reductions in SNS-induced PADs. Combined applications of these blockers led to little or no further repression of SNS-induced PADs, suggesting interactions among A(2)Rs and K(+) channels. In the absence of SNS, BaCl(2) blockade of K(ir) channels produced 52-80% reductions in Ado and NS-1619 (BK(Ca) channel activator)-induced PADs. In contrast, paxilline blockade of BK(Ca) channels was without effect on dilations elicited by KCl (K(ir) channel activator) and Ado suffusions, indicating that Ado- and NS-1619-associated PADs involved K(ir) channels. In addition, targeted ablation of the superficial glia limitans was associated with a selective 60-80% loss of NS-1619 responses, suggesting that the BK(Ca) channel participation (and paxilline sensitivity) derived largely from channels within the glia limitans. Additionally, blockade of either PKA or adenylyl cyclase caused markedly attenuated pial arteriolar responses to SNS and, in the absence of SNS, responses to Ado, KCl, and NS-1619. These findings suggested a key, possibly permissive, role for A(2)R-linked cAMP generation and PKA-induced K(+) channel phosphorylation in somatosensory activation-evoked PAD.     

Bromine patterns in Norwegian coastal Cod otoliths—a possible marker for distinguishing stocks?

Bromine was found to accumulate in otoliths of Norwegian coastal Cod Gadus morhua that were reared under known conditions. Despite the fact that the Cod were moved from one rearing environment to another, causing marked changes in some otolith elemental concentrations, bromine appeared to accumulate continuously along certain growth axes as revealed by 2-D elemental mapping. In contrast, North Sea and Baltic Sea Cod showed little to no patterning in Br. We suggest that Br uptake in otoliths may be under physiological and genetic control, and as such, may prove useful as a stock identification tool.     

Stratospheric microbiology at 20 km over the Pacific Ocean

An aerobiology sampling flight at 20 km was conducted on 28 April 2008 over the Pacific Ocean (36.5° N, 118–149° W), a period of time that coincided with the movement of Asian dust across the ocean. The aim of this study was to confirm the presence of viable bacteria and fungi within a transoceanic, atmospheric bridge and to improve the resolution of flight hardware processing techniques. Isolates of the microbial strains recovered were analyzed with ribosomal ribonucleic acid (rRNA) sequencing to identify bacterial species Bacillus sp., Bacillus subtilis, Bacillus endophyticus, and the fungal genus Penicillium. Satellite imagery and ground-based radiosonde observations were used to measure dust movement and characterize the high-altitude environment at the time of collection. Considering the atmospheric residency time (7–10 days), the extreme temperature regime of the environment (−75°C), and the absence of a mechanism that could sustain particulates at high altitude, it is unlikely that our samples indicate a permanent, stratospheric ecosystem. However, the presence of viable fungi and bacteria in transoceanic stratosphere remains relevant to understanding the distribution and extent of microbial life on Earth.     

Differential risk effects of wolves on wild versus domestic prey have consequences for conservation

Predators play integral roles in shaping ecosystems through cascading effects to prey and vegetation. Such effects occur when prey species alter their behavior to avoid predators, a phenomenon called the risk effects of predators. Risk effects of wild predators such as wolves are well documented for wild prey, but not for free ranging domestic animals such as cattle despite their importance for ecosystem function and conservation. We compared risk effects of satellite‐collared wolves (n = 16) on habitat selection by global‐positioning‐system‐collared elk (n = 10) and cattle (n = 31). We calculated resource selection functions (RSFs) in periods before, during and after wolf visits in elk home ranges or cattle pastures. The habitat variables tested included: distance to roads and trails, terrain ruggedness, food‐quality and distance to forest. When wolves were present, elk stayed closer to forest cover and selected less for high‐quality‐food habitat. Thus, the risk effects of wolf presence on elk produced a change in the tradeoff between food and cover selection. Cattle responded by avoiding high‐quality‐food habitat and selecting areas closer to roads and trails (where people likely provided security), but these effects manifested only after wolves had left. Artificial selection in cattle may have attenuated natural anti‐predator behaviors. The effects of predators on ecosystems are likely different when mediated through risk effects on domestic compared to wild animals. Furthermore, predator control in response to livestock predation, an important conservation issue, may produce broad ecosystem effects triggered by decrease of an important predator species. Conservation planners should consider these effects where domestic herbivores are dominant species in the ecosystem.     

Polyphasic Characterization of a Thermotolerant Siderophilic Filamentous Cyanobacterium That Produces Intracellular Iron Deposits

Despite the high potential for oxidative stress stimulated by reduced iron, contemporary iron-depositing hot springs with circum-neutral pH are intensively populated with cyanobacteria. Therefore, studies of the physiology, diversity, and phylogeny of cyanobacteria inhabiting iron-depositing hot springs may provide insights into the contribution of cyanobacteria to iron redox cycling in these environments and new mechanisms of oxidative stress mitigation. In this study the morphology, ultrastructure, physiology, and phylogeny of a novel cyanobacterial taxon, JSC-1, isolated from an iron-depositing hot spring, were determined. The JSC-1 strain has been deposited in ATCC under the name Marsacia ferruginose, accession number BAA-2121. Strain JSC-1 represents a new operational taxonomical unit (OTU) within Leptolyngbya sensu lato. Strain JSC-1 exhibited an unusually high ratio between photosystem (PS) I and PS II, was capable of complementary chromatic adaptation, and is apparently capable of nitrogen fixation. Furthermore, it synthesized a unique set of carotenoids, but only chlorophyll a. Strain JSC-1 not only required high levels of Fe for growth (≥40 μM), but it also accumulated large amounts of extracellular iron in the form of ferrihydrite and intracellular iron in the form of ferric phosphates. Collectively, these observations provide insights into the physiological strategies that might have allowed cyanobacteria to develop and proliferate in Fe-rich, circum-neutral environments.Cyanobacteria inhabiting ferrous iron-rich hot springs with circum-neutral pH represent unique models for examining the mechanisms by which early organisms evolved to cope with such habitats common on early Earth. Such organisms have previously been shown to be resistant to Fe²⁺ (37) or Fe³⁺ (6, 7) at concentrations in the micromolar to millimolar range. Moreover, high Fe concentrations (apparent optimum of ∼0.5 mM) stimulated the growth of these cyanobacteria, which were described as siderophilic (having an affinity for iron) cyanobacteria (7).The cyanobacteria inhabiting the Chocolate Pots hot springs in Yellowstone National Park, Wyoming, were shown to have played at least a passive role in contributing to iron deposition by serving as nucleation sites for the accumulation of iron minerals and associated silica deposits (36, 38). The precipitation of external iron that encrusts the cyanobacterial cells inhabiting this hot spring appears to be dependent on the species composition and chemistry of the mat (36, 38); however, multiple anoxygenic phototrophs found in the Chocolate Pots hot springs (8) could also contribute to the formation of Fe oxides (21, 49). Therefore, only iron mineralization experiments with model cyanobacterial strains can demonstrate the role of siderophilic cyanobacteria in the formation of specific, iron-bearing minerals.An additional common feature of circum-neutral iron-depositing hot springs is elevated concentrations of hydrogen peroxide (50). Shcolnick and coauthors (41) showed that a wild type of Synechococcus sp. PCC 6803 was resistant to 8 mM H₂O₂ if grown with 0.3 μM Fe³⁺, while the same concentration of hydrogen peroxide completely inhibited the growth of this cyanobacterium if it was grown with 10 μM Fe³⁺. If a similar correlation between iron concentration and the magnitude of an externally applied oxidative stress were the case for siderophilic cyanobacteria, iron-depositing hot springs should be free of cyanobacteria. However, such springs are very rich with cyanobacteria (38, 7, 36), which suggests that siderophilic cyanobacteria may possess unusual mechanisms of iron homeostasis maintenance and oxidative stress mitigation. Additionally, understanding iron tolerance and phenomena associated with siderophily in oxygenic prokaryotes is also important because such siderophilic organisms might help us find applications for bioremediation of waters polluted with iron.The current work describes the morphology, ultrastructure, physiology, and phylogeny of a previously undescribed, siderophilic cyanobacterium. The results of this polyphasic characterization led to the conclusion that strain JSC-1 represents a new operational taxonomic unit (OTU). (The epithet for JSC-1, Marsacia ferruginose, was chosen in honor of Nicole Tandeau de Marsac.) Additionally, biomineralization of intracellular iron by a cyanobacterium is demonstrated for the first time.     

The discovery of a series of N-substituted 3-(4-piperidinyl)-1,3-benzoxazolinones and oxindoles as highly brain penetrant,selective muscarinic M1 agonists

A series of N-substituted 3-(4-piperidinyl)-1,3-benzoxazolinones and oxindoles are reported which were found to be potent and selective muscarinic M₁ agonists. By control of the physicochemical characteristics of the series, particularly the lipophilicity, compounds with good metabolic stability and excellent brain penetration were identified. An exemplar of the series was shown to be pro-cognitive in the novel object recognition rat model of temporal induced memory deficit.     

Studies on the structure–activity relationship of 1,3,3,4-tetra-substituted pyrrolidine embodied CCR5 receptor antagonists. Part 2: Discovery of highly potent anti-HIV agents

Modification of 1,3,3,4-tetra-substituted pyrrolidine embodied CCR5 receptor antagonists revealed that introducing a fluoro group at the 3-position of the 3-phenyl group to reduce metabolism did not adversely affect the high potency against HIV infection, and that replacing the piperidine ring with a tropane ring could deliver the most potent anti-HIV agents. Stereochemistry of the substituted tropane ring is essential for maintaining the potent anti-HIV activity because only exo-isomers displayed subnanomolar whole cell activity.     

Breaking the covalent connection: Chain connectivity and the catalytic reaction of PMM/PGM

Fragment complementation has been used to investigate the role of chain connectivity in the catalytic reaction of phosphomannomutase/phosphoglucomutase (PMM/PGM) from Pseudomonas aeruginosa, a human pathogen. A heterodimer of PMM/PGM, created from fragments corresponding to its first three and fourth domains, was constructed and enzyme activity reconstituted. NMR spectra demonstrate that the fragment corresponding to the fourth (C‐terminal) domain exists as a highly structured, independent folding domain, consistent with its varied conformation observed in enzyme–substrate complexes. Steady‐state kinetics and thermodynamics studies reported here show that complete conformational freedom of Domain 4, because of the break in the polypeptide chain, is deleterious to catalytic efficiency primarily as a consequence of increased entropy. This extends observations from studies of the intact enzyme, which showed that the degree of flexibility of a hinge region is controlled by the precise sequence of amino acids optimized through evolutionary constraints. This work also sheds light on the functional advantage gained by combining separate folding domains into a single polypeptide chain.     

<Emphasis Type="Italic">In vitro</Emphasis> and <Emphasis Type="Italic">ex vivo</Emphasis> effect of hyaluronic acid on erythrocyte flow properties

Background  

Hyaluronic acid (HA) is present in many tissues; its presence in serum may be related to certain inflammatory conditions, tissue damage, sepsis, liver malfunction and some malignancies. In the present work, our goal was to investigate the significance of hyaluronic acid effect on erythrocyte flow properties. Therefore we performed in vitro experiments incubating red blood cells (RBCs) with several HA concentrations. Afterwards, in order to corroborate the pathophysiological significance of the results obtained, we replicated the in vitro experiment with ex vivo RBCs from diagnosed rheumatoid arthritis (RA) patients, a serum HA-increasing pathology.