First microwave-assisted synthesis of an electron-rich phosphane and its coordination chemistry with platinum(II) and palladium(II)

The P-O ligand 3-(di(2-methoxyphenyl)phosphanyl)propionic acid (HL) was synthesized by a microwave-assisted reaction of a secondary phosphane. The coordination of HL to Pt(II) yielded the neutral mononuclear complex trans-[PtCl(κ(2)-P,O-L)(κ-P-HL)] (1), while the reaction of PdClMe(η(4)-COD) (COD?=?1,4-cyclooctadiene) with HL in the presence of NEt(3) gave the anionic Pd(II) compound of the formula (HNEt(3))[PdClMe(κ(2)-P,O-L)] (2). Upon crystallization of the latter compound the neutral chloride-bridged dimetallic compound cis-[Pd(μ-Cl)Me(HL)](2) (3) was obtained. HL, 1 and 3·CH(2)Cl(2) have been characterized by single crystal X-ray structure analyses.     

The exocytotic fusion pore modeled as a lipidic pore.

Freeze-fracture electron micrographs from degranulating cells show that the lumen of the secretory granule is connected to the extracellular compartment via large (20 to 150 nm diameter) aqueous pores. These exocytotic fusion pores appear to be made up of a highly curved bilayer that spans the plasma and granule membranes. Conductance measurements, using the patch-clamp technique, have been used to study the fusion pore from the instant it conducts ions. These measurements reveal the presence of early fusion pores that are much smaller than those observed in electron micrographs. Early fusion pores open abruptly, fluctuate, and then either expand irreversibly or close. The molecular structure of these early fusion pores is unknown. In the simplest extremes, these early fusion pores could be either ion channel like protein pores or lipidic pores. Here, we explored the latter possibility, namely that of the early exocytotic fusion pore modeled as a lipid-lined pore whose free energy was composed of curvature elastic energy and work done by tension. Like early exocytotic fusion pores, we found that these lipidic pores could open abruptly, fluctuate, and expand irreversibly. Closure of these lipidic pores could be caused by slight changes in lipid composition. Conductance distributions for stable lipidic pores matched those of exocytotic fusion pores. These findings demonstrate that lipidic pores can exhibit the properties of exocytotic fusion pores, thus providing an alternate framework with which to understand and interpret exocytotic fusion pore data.     

Ecological niches in sequential generations of eastern North American monarch butterflies (Lepidoptera: Danaidae): the ecology of migration and likely climate change implications

Eastern North American monarch butterflies (Danaus plexippus L.) show a series of range shifts during their breeding season. Using ecological niche modeling, we studied the environmental context of these shifts by identifying the ecological conditions that monarchs use in successive summer months. Monarchs use a consistent ecological regimen through the summer, but these conditions contrast strikingly with those used during the winter. Hence, monarchs exhibit niche-following among sequential breeding generations but niche-switching between the breeding and overwintering stages of their annual cycle. We projected their breeding ecological niche onto monthly future climate scenarios, which indicated northward shifts, particularly at the northern extreme of their summer movements, over the next 50 yrs; if both monarchs and their milkweed host plants cannot track these changing climates, monarchs could lose distributional area during critical breeding months.     

Simultaneous capacitance and amperometric measurements of exocytosis: a comparison.

We measured the exocytotic response induced by flash photolysis of caged compounds in isolated mast cells and chromaffin cells. Vesicle fusion was measured by monitoring the cell membrane capacitance. The release of vesicular contents was followed by amperometry. In response to a GTP gamma S stimulus we found that the time integral of the amperometric current could be superimposed on the capacitance trace. This shows that the integrated amperometric signal provides an alternative method of measuring the extent and kinetics of the secretory response. Very different results were obtained when photolysis of caged Ca2+ (DM-nitrophen) was used to stimulate secretion. In mast cells, there was an immediate, graded increase in membrane capacitance that was followed by step increases (indicative of granule fusion). During the initial phase of the capacitance increases, no release of oxidizable secretory products was detected. In chromaffin cells we also observed a considerable delay between increases in capacitance, triggered by uncaging Ca2+, and the release of oxidizable secretory products. Here we demonstrate that there can be large increases in the membrane capacitance of a secretory cell, triggered by flash photolysis of DM-nitrophen, which indicate events that are not due to the fusion of granules containing oxidizable substances. These results show that increases in capacitance that are not resolved as steps cannot be readily interpreted as secretory events unless they are confirmed independently.     

Naturally occurring osmolytes modulate the nanomechanical properties of polycystic kidney disease domains

Polycystin-1 (PC1) is a large membrane protein that is expressed along the renal tubule and exposed to a wide range of concentrations of urea. Urea is known as a common denaturing osmolyte that affects protein function by destabilizing their structure. However, it is known that the native conformation of proteins can be stabilized by protecting osmolytes that are found in the mammalian kidney. PC1 has an unusually long ectodomain with a multimodular structure including 16 Ig-like polycystic kidney disease (PKD) domains. Here, we used single-molecule force spectroscopy to study directly the effects of several naturally occurring osmolytes on the mechanical properties of PKD domains. This experimental approach more closely mimics the conditions found in vivo. We show that upon increasing the concentration of urea there is a remarkable decrease in the mechanical stability of human PKD domains. We found that protecting osmolytes such as sorbitol and trimethylamine N-oxide can counteract the denaturing effect of urea. Moreover, we found that the refolding rate of a structurally homologous archaeal PKD domain is significantly slowed down in urea, and this effect was counteracted by sorbitol. Our results demonstrate that naturally occurring osmolytes can have profound effects on the mechanical unfolding and refolding pathways of PKD domains. Based on these findings, we hypothesize that osmolytes such as urea or sorbitol may modulate PC1 mechanical properties and may lead to changes in the activation of the associated polycystin-2 channel or other intracellular events mediated by PC1.     

Species distribution models for natural enemies of monarch butterfly (<Emphasis Type="Italic">Danaus plexippus</Emphasis>) larvae and pupae: distribution patterns and implications for conservation

Prey populations can be strongly influenced by predators and parasitoids, and migratory prey whose distributions vary geographically throughout their breeding seasons encounter different combinations of predators and parasitoids throughout their range. North American monarch butterflies (Danaus plexippus) are susceptible to a wide variety of natural enemies, but the distribution of these natural enemies has not been quantified. We developed ecological niche models using environmental data to identify areas with suitable abiotic conditions for eight known natural enemies of monarchs, including six predators: Arilus cristatus, Harmonia axyridis, Monomorium minimum, Podisus maculiventris, Polistes spp., and Solenopsis geminata; and two parasitoids: Lespesia archippivora and Pteromalus cassotis. We combined correlated suitable areas for individual predators and parasitoids to identify regions with the most predator and parasitoid species potential. The Gulf Coast, West Coast, Florida, and parts of the eastern United States are predicted to have the most natural enemy species. We suggest that future research should assess monarch mortality rates in these areas, and that monarch conservation strategies consider pressure from natural enemies.     

Role of Wnt-5a in the Determination of Human Mesenchymal Stem Cells into Preadipocytes

Increasing adipocyte size as well as numbers is important in the development of obesity and type 2 diabetes, with adipocytes being generated from mesenchymal precursor cells. This process includes the determination of mesenchymal stem cells (MSC) into preadipocytes (PA) and the differentiation of PA into mature fat cells. Although the process of differentiation has been highly investigated, the determination in humans is poorly understood. In this study, we compared human MSC and human committed PA on a cellular and molecular level to gain further insights into the regulatory mechanisms in the determination process. Both cell types showed similar morphology and expression patterns of common mesenchymal and hematopoietic surface markers. However, although MSC were able to differentiate into adipocytes and osteocytes, PA were only able to undergo adipogenesis, indicating that PA lost their multipotency during determination. WNT-5a expression showed significantly higher levels in MSC compared with PA suggesting that WNT-5a down-regulation might be important in the determination process. Indeed, incubation of human MSC in medium containing neutralizing WNT-5a antibodies abolished their ability to undergo osteogenesis, although adipogenesis was still possible. An opposite effect was achieved using recombinant WNT-5a protein. On a molecular level, WNT-5a was found to promote c-Jun N-terminal kinase-dependent intracellular signaling in MSC. Activation of this noncanonical pathway resulted in the induction of osteopontin expression further indicating pro-osteogenic effects of WNT-5a. Our data suggest that WNT-5a is necessary to maintain osteogenic potential of MSC and that inhibition of WNT-5a signaling therefore plays a role in their determination into PA in humans.     

Weight loss due to a very low calorie diet differentially affects insulin sensitivity and interleukin-6 serum levels in nondiabetic obese human subjects

Inflammatory mechanisms are involved in the pathogenesis of type 2 diabetes with interleukin (IL)-6 being particularly important. While long term exercise has been shown to be associated with reduction in IL-6 serum levels in several reports, the discussion on the effect of dietary intervention on IL-6 serum levels is controversial. In the present study, we aimed to investigate the effect of weight loss due to a very low calorie diet (VLCD) on insulin sensitivity and IL-6 serum levels in nondiabetic obese human individuals. 10 patients with obesity were examined during 12 weeks of a VLCD (800 kcal/d). Body composition was measured by impedance analysis. Blood samples were taken before, during, and after the dietary intervention. Leptin, adiponectin, and IL-6 serum levels were measured by ELISA. The body weight decreased significantly from 123.9±6.2-103.5±5.6 kg with a significant reduction in body fat content (43.2±2.3-36.1±3.1%). Leptin levels exhibited a significant decrease from 56.8±5.6-27.9±5.6 ng/ml while adiponectin levels increased significantly from 7.5±0.9-10.6±1.1 μg/ml. Thereby the leptin-to-adiponectin ratio, a novel marker for insulin sensitivity, significantly improved. Mean IL-6 serum concentrations were within the normal range (3.2±0.8 pg/ml) before the study and were not significantly altered by the nutritional therapy. Despite improvement of insulin sensitivity, IL-6 serum levels did not change throughout the study period, suggesting that in nondiabetic obese human subjects IL-6 might have only a minor role in the impairment of insulin sensitivity.     

Single-molecule force spectroscopy reveals a stepwise unfolding of Caenorhabditis elegans giant protein kinase domains

Myofibril assembly and disassembly are complex processes that regulate overall muscle mass. Titin kinase has been implicated as an initiating catalyst in signaling pathways that ultimately result in myofibril growth. In titin, the kinase domain is in an ideal position to sense mechanical strain that occurs during muscle activity. The enzyme is negatively regulated by intramolecular interactions occurring between the kinase catalytic core and autoinhibitory/regulatory region. Molecular dynamics simulations suggest that human titin kinase acts as a force sensor. However, the precise mechanism(s) resulting in the conformational changes that relieve the kinase of this autoinhibition are unknown. Here we measured the mechanical properties of the kinase domain and flanking Ig/Fn domains of the Caenorhabditis elegans titin-like proteins twitchin and TTN-1 using single-molecule atomic force microscopy. Our results show that these kinase domains have significant mechanical resistance, unfolding at forces similar to those for Ig/Fn β-sandwich domains (30-150 pN). Further, our atomic force microscopy data is consistent with molecular dynamic simulations, which show that these kinases unfold in a stepwise fashion, first an unwinding of the autoinhibitory region, followed by a two-step unfolding of the catalytic core. These data support the hypothesis that titin kinase may function as an effective force sensor.