Substitution, abstraction and addition chemistry of low-coordinate gallium and indium ligand systems

Substitution, abstraction and addition processes have been shown to be viable chemistries for the modification of ligand systems featuring heavier group 13 element donor atoms. Thus substitution of the bromide in Cp^∗Fe(CO)₂In(Br)Mes^∗ (1) can be carried out with retention of the Fe-In bond, using 1 equiv. of the aryloxide nucleophile [OC₆H₄^tBu-4]⁻ to give Cp^∗Fe(CO)₂In(OC₆H₄^tBu-4)Mes^∗ (2). Structural and spectroscopic comparisons of 1 and 2 reveal that variation in the steric and/or π donor properties of the indyl ligand substituents have little effect on the nature of the Fe-In bond. Sequential reaction of [Cp^∗Fe(CO)₂]₂GaCl (3) with the halide abstraction agent and 4-picoline in dichloromethane proceeds via the known two-coordinate gallium cation (4). The net result is replacement of the gallium bound chloride substituent with a 4-picoline moiety, yielding (5) via a two-step abstraction/addition process. 5 represents only the second structurally characterized complex containing a cationic three-coordinate gallium centre, and the first displaying bonds to a transition metal.     

Peripheral ghrelin deepens torpor bouts in mice through the arcuate nucleus neuropeptide Y signaling pathway

Many small mammals have the ability to enter torpor, characterized by a controlled drop in body temperature (Tb). We hypothesized that ghrelin would modulate torpor bouts, because torpor is induced by fasting in mice coincident with elevated circulating ghrelin. Female National Institutes of Health (NIH) Swiss mice were implanted with a Tb telemeter and housed at an ambient temperature (Ta) of 18 degrees C. On fasting, all mice entered a bout of torpor (minimum Tb: 23.8+/-2.0 degrees C). Peripheral ghrelin administration (100 microg) during fasting significantly deepened the bout of torpor (Tb minimum: 19.4+/-0.5 degrees C). When the arcuate nucleus (ARC) of the hypothalamus, a ghrelin receptor-rich region of the brain, was chemically ablated with monosodium glutamate (MSG), fasted mice failed to enter torpor (minimum Tb=31.6+/-0.6 degrees C). Furthermore, ghrelin administration had no effect on the Tb minimum of ARC-ablated mice (31.8+/-0.8 degrees C). Two major pathways that regulate food intake reside in the ARC, the anorexigenic alpha-melanocyte stimulating hormone (alpha-MSH) pathway and the orexigenic neuropeptide Y (NPY) signaling pathway. Both Ay mice, which have the alpha-MSH pathway blocked, and Npy-/-mice exhibited shallow, aborted torpor bouts in response to fasting (Tb minimum: 29.1+/-0.6 degrees C and 29.9+/-1.2 degrees C, respectively). Ghrelin deepened torpor in Ay mice (Tb minimum: 22.8+/-1.3 degrees C), but had no effect in Npy-/-mice (Tb minimum: 29.5+/-0.8 degrees C). Collectively, these data suggest that ghrelin's actions on torpor are mediated via NPY neurons within the ARC.     

Neutrophil activation is modulated by sex hormones after trauma-hemorrhagic shock and burn injuries

Recent literature indicates that females are more resistant to shock-, trauma-, and sepsis-induced immune dysfunction and organ injury than are males. Consequently, using trauma-hemorrhagic shock (T/HS) and burn models, we tested whether the neutrophil response to trauma occurred in a sexually dimorphic fashion and, if so, the role of sex hormones. Neutrophil activation, as reflected by CD11b expression and respiratory burst activity, was increased to a greater extent in male rats than in female rats after T/HS or burn injury. Testosterone appeared to potentiate neutrophil activation, because castration reduced neutrophil activation, whereas ovariectomy had little effect. Mechanistically, this sexually dimorphic neutrophil response appeared to be due to both cellular and humoral factors. Evidence for a cellular difference between male and female neutrophils is based on the observation that naive female neutrophils were more resistant to activation by burn or T/HS plasma and lymph than naive male neutrophils and that this resistance varied over the estrus cycle. Additionally, the humoral environment was more neutrophil activating in male rats, because burn and T/HS plasma and lymph from male rats activated naive male neutrophils to a greater extent than comparable samples from females. Last, on the basis of in vitro experiments examining the effects of estrogen on calcium signaling, it appears that estrogen limits trauma-induced neutrophil activation, at least in part, by limiting the entry of calcium into the cell via store-operated calcium entry mechanisms. In conclusion, there is a striking sexual dimorphism in neutrophil responses after trauma, and these changes reflect both cellular resistance to activation as well as a less activating humoral environment.     

Ligand-independent regulation of ErbB4 receptor phosphorylation by activated Ras

The ErbB family of receptor tyrosine kinases regulates cell growth, differentiation and survival. Activation of the receptors is induced by specific growth factors in an autocrine, paracrine or juxtacrine manner. The activated ErbB receptors turn on a large variety of signaling cascades, including the prominent Ras-dependent signaling pathways. The activated Ras can induce secretion of growth factors such as EGF and neuregulin, which activate their respective receptors. In the present study, we demonstrate for the first time that activated Ras can activate ErbB4 receptor in a ligand-independent manner. Expression of constitutively active H-Ras(12V), K-Ras(12V) or N-Ras(13V) in PC12-ErbB4 cells induced ErbB4-receptor phosphorylation, indicating that each of the most abundant Ras isoforms can induce receptor activation. NRG-induced phosphorylation of ErbB4 receptor was blocked by the soluble ErbB4 receptor, which had no effect on the Ras-induced receptor phosphorylation. Moreover, conditioned medium from H-Ras(12V)-transfected PC12-ErbB4 cells had no effect on receptor phosphorylation. It thus indicates that Ras induces ErbB4 phosphorylation in a ligand-independent manner. Each of the Ras effector domain mutants, H-Ras(12V)S35, H-Ras(12V)C40, and H-Ras(12V)G37, which respectively activate Raf1, PI3K, and RalGEF, induced a small but significant receptor phosphorylation. The PI3K inhibitor LY294002 and the MEK inhibitor PD98059 caused a partial inhibition of the Ras-induced ErbB4 receptor phosphorylation. Using a mutant ErbB4 receptor, which lacks kinase activity, we demonstrated that the Ras-mediated ErbB4 phosphorylation depends on the kinase activity of the receptor and facilitates ligand-independent neurite outgrowth in PC12-ErbB4 cells. These experiments demonstrate a novel mechanism controlling ErbB receptor activation. Ras induces ErbB4 receptor phosphorylation in a non-autocrine manner and this activation depends on multiple Ras effector pathways and on ErbB4 kinase activity.     

An empirical verification of population assignment methods by marking and parentage data: hatchery and wild steelhead (Oncorhynchus mykiss) in Forks Creek, Washington, USA

Assignment tests are increasingly applied in ecology and conservation, although empirical comparisons of methods are still rare or are restricted to few of the available approaches. Furthermore, the performance of assignment tests in cases with low population differentiation, violations of Hardy-Weinberg equilibrium and unbalanced sampling designs has not been verified. The release of adult hatchery steelhead to spawn in Forks Creek in 1996 and 1997 provided an opportunity to compare the power of different assignment methods to distinguish their offspring from those of sympatric wild steelhead. We compared standard assignment methods requiring baseline samples (frequency, distance and Bayesian) and clustering approaches with and without baseline information, using six freely available computer programs. Assignments were verified by parentage data obtained for a subset of returning offspring. All methods provided similar assignment success, despite low differentiation between wild and hatchery fish (F(ST) = 0.02). Bayesian approaches with baseline data performed best, whereas the results of clustering methods were variable and depended on the samples included in the analysis and the availability of baseline information. Removal of a locus with null alleles and equalizing sample sizes had little effect on assignments. Our results demonstrate the robustness of most assignment tests to low differentiation and violations of assumptions, as well as their utility for ecological studies that require correct classification of different groups.     

GENETIC POPULATION STRUCTURE OF PSEUDO‐NITZSCHIA PUNGENS (BACILLARIOPHYCEAE) FROM THE PACIFIC NORTHWEST AND THE NORTH SEA1

Several species of the diatom Pseudo‐nitzschia produce the neurotoxin domoic acid (DA). Consumption of fish and shellfish that have accumulated this potent excitotoxin has resulted in severe illness and even death in humans, marine mammals, and seabirds. Pseudo‐nitzschia pungens (Grunow ex Cleve) Hasle is a cosmopolitan diatom commonly occurring in the waters of the Pacific Northwest (PNW) and the eastern North Atlantic, including the North Sea. However, genetic and physiological relationships among populations throughout this large geographic distribution have not been assessed. Population genetic parameters (e.g., Hardy–Weinberg equilibrium, linkage equilibrium, F_ST) calculated for P. pungens collected from the Juan de Fuca eddy region in the PNW indicated the presence of two distinct groups that were more divergent from each other than either was from a P. pungens sample from the North Sea. Geographic heterogeneity was also detected within each of the two PNW groups. These results suggested that the populations of P. pungens recently mixed in the Juan de Fuca eddy region (a seasonally retentive feature off the coasts of Washington State, USA, and Vancouver Island, Canada) but did not exchange genetic material by sexual reproduction. Alternatively, these two groups may be cryptic (morphologically identical, but reproductively isolated) species. Identifying cryptic diversity in Pseudo‐nitzschia is important for bloom prediction and aiding the identification of molecular markers that can be used for rapid detection assay development.     

ANALYSIS OF A PLASTID MULTIGENE DATA SET AND THE PHYLOGENETIC POSITION OF THE MARINE MACROALGA CAULERPA FILIFORMIS (CHLOROPHYTA)1

Molecular phylogenetic relationships within the Chlorophyta have relied heavily on rRNA data. These data have revolutionized our insight in green algal evolution, yet some class relationships have never been well resolved. A commonly used class within the Chlorophyta is the Ulvophyceae, although there is not much support for its monophyly. The relationships among the Ulvophyceae, Trebouxiophyceae, and Chlorophyceae are also contentious. In recent years, chloroplast genome data have shown their utility in resolving relationships between the main green algal clades, but such studies have never included marine macroalgae. We provide partial chloroplast genome data (～30,000 bp, 23 genes) of the ulvophycean macroalga Caulerpa filiformis (Suhr) K. Herig. We show gene order conservation for some gene combinations and rearrangements in other regions compared to closely related taxa. Our data also revealed a pseudogene (ycf62) in Caulerpa species. Our phylogenetic results, based on analyses of a 23‐gene alignment, suggest that neither Ulvophyceae nor Trebouxiophyceae are monophyletic, with Caulerpa being more closely related to the trebouxiophyte Chlorella than to Oltmannsiellopsis and Pseudendoclonium.     

Potent and cellularly active 4-aminoimidazole inhibitors of cyclin-dependent kinase 5/p25 for the treatment of Alzheimer’s disease

Utilizing structure-based drug design, a 4-aminoimidazole heterocyclic core was synthesized as a replacement for a 2-aminothiazole due to potential metabolically mediated toxicity. The synthetic route utilized allowed for ready synthesis of 1-substituted-4-aminoimidazoles. SAR exploration resulted in the identification of a novel cis-substituted cyclobutyl group that gave improved enzyme and cellular potency against cdk5/p25 with up to 30-fold selectivity over cdk2/cyclin E.     

Substrate specificity effects of lipoxygenase products and inhibitors on soybean lipoxygenase-1

Recently, it has been shown that lipoxygenase (LO) products affect the substrate specificity of human 15-LO. In the current paper, we demonstrate that soybean LO-1 (sLO-1) is not affected by its own products, however, inhibitors which bind the allosteric site, oleyl sulfate (OS) and palmitoleyl sulfate (PS), not only lower catalytic activity, but also change the substrate specificity, by increasing the arachidonic acid (AA)/linoleic acid (LA) ratio to 4.8 and 4.0, respectively. The fact that LO inhibitors can lower activity and also change the LO product ratio is a new concept in lipoxygenase inhibition, where the goal is to not only reduce the catalytic activity but also alter substrate selectivity towards a physiologically beneficial product.