Genistein inhibits PDGF-stimulated proteoglycan synthesis in vascular smooth muscle without blocking PDGFβ receptor phosphorylation

The signaling pathways that regulate the synthesis and structure of proteoglycans secreted by vascular smooth muscle cells are potential therapeutic targets for preventing lipid deposition in the early stage of atherosclerosis. PDGF stimulates both core protein expression and elongation of glycosaminoglycan (GAG) chains on proteoglycans. In this study we investigated the effects of the tyrosine kinase inhibitor genistein on PDGF mediated receptor phosphorylation and proteoglycan synthesis in human vascular smooth muscle cells. We demonstrate that genistein does not block phosphorylation of the activation site of the PDGF receptor at Tyr(857) and two other downstream sites Tyr(751) and Tyr(1021). Genistein blocked PDGF-mediated proteoglycan core protein synthesis however it had no effect on GAG chain elongation. These results differ markedly to two other tyrosine kinase inhibitors, imatinib and Ki11502, that block PDGF receptor phosphorylation and PDGF mediated GAG elongation. We conclude that the action of genistein on core protein synthesis does not involve the PDGF receptor and that PDGF mediates GAG elongation via the PDGF receptor.     

Pollen stratigraphy of coal-bearing deposits in the Neogene Jidong Basin,Heilongjiang Province,NE China: New insights on palaeoenvironment and age

The palaeoenvironment and age of coal-bearing lacustrine deposits in the Jidong Basin, Heilongjiang Province, is poorly understood. New pollen data from the Yongqing Formation and the overlying lower part of the Daqingshan Formation recovered in borehole 91-205 revealed a rich and diverse palynoflora representing approximately 70 taxa at the genus/family level. Of these taxa, Ulmus, Quercus, Carya, Liquidambar, Pinus, Tilia, Fagus, and Alnus are the main woody elements; these occur together with numerous pteridophytes, subordinate herbs, and rare algae and acritarchs. Abundant woody taxa suggest a mixed deciduous/conifer forest in the region in association with local shrubs, herbs and algal communities. Some of the pollen types, especially those of climate sensitive genera such as Ulmus, Carya, Liquidambar and Fagus, vary considerably in relative abundances, suggesting climate variability. The reflected climate shows an overall trend towards cooling or deterioration which is largely in agreement with global climate change during the Neogene.Comparisons of the pollen record with representative datasets from neighbouring areas, together with the appearance of the highly-evolved genus Artemisia, suggest that the Yongqing Formation and the lower part of the Daqingshan Formation are of Miocene age.     

Developmental toxicity of artesunate in the rat: comparison to other artemisinins, comparison of embryotoxicity and kinetics by oral and intravenous routes, and relationship to maternal reticulocyte count

BACKGROUND: The antimalarial, artesunate, is teratogenic and embryolethal in rats, with peak sensitivity on Days 10 and 11 postcoitum (pc). METHODS: We compared the developmental toxicity of structurally related artemisinins, dihdyroartemisinin (DHA), artemether (ARTM), and arteether (ARTE) to that of artesunate after oral administration to rats on Day 10 pc. In separate studies, embryolethality was characterized after single intravenous (IV) administration of artesunate on Day 11 pc, and toxicokinetic parameters following oral and IV administration were compared. Lastly, to determine whether maternal hematologic effects occurred at doses that affect embryonic erythroblasts, artesunate was orally administered on Day 11 pc at a dose that caused 100% embryolethality. RESULTS: All artemisinins caused the same pattern of embryolethality and fetal cardiovascular and skeletal abnormalities as previously shown for artesunate. In the IV study, marked postimplantation loss occurred at 1.5 and 3 mg/kg artesunate, but not at 0.75 mg/kg. Among the toxicokinetic parameters evaluated, only the DHA AUC_0‐t was similar at embryolethal oral and IV doses of artesunate. An embryolethal dose of artesunate caused a 15% decrease in maternal reticulocyte counts and no other hematologic effects. CONCLUSIONS: Several structurally related artemisinins cause similar developmental toxicity, suggesting an artemisinin class effect. Equally embryotoxic oral and IV treatments of one artemisinin compound (artesunate) produced similar systemic exposure to the artesunate metabolite, DHA, suggesting that DHA may be the proximate developmental toxicant. Embryolethal doses of artesunate only caused minor changes in maternal reticulocyte counts indicating that adult hematology parameters are not as sensitive as embryonic erythroblasts. Birth Defects Res (Part B) 83:397–406, 2008. © 2008 Wiley‐Liss, Inc.     

Regulation of cell–cell adhesion during Dictyostelium development

During development of Dictyostelium, four adhesion systems have been identified and adherens junction-like structures have been discovered in the fruiting body. The temporal and spatial expression of cell adhesion molecules (CAMs) is under stringent developmental control, corresponding to major shifts in morphological complexity. Genetic manipulations, including over-expression and knockout mutations, of the adhesion genes, cadA (encoding DdCAD-1), csaA (gp80) and lagC (gp150), have shed light on new roles for cell adhesion molecules in aggregate size regulation, cell-type proportioning, cell differentiation and cell sorting. As cell–cell interactions remain highly dynamic within cell streams and aggregates, mechanisms must exist to facilitate the rapid assembly and disassembly of adhesion complexes. Studies on gp80 have led to a model for the rapid assembly of adhesion complexes via lipid rafts.     

A predominantly neolithic origin for Y-chromosomal DNA variation in North Africa

We have typed 275 men from five populations in Algeria, Tunisia, and Egypt with a set of 119 binary markers and 15 microsatellites from the Y chromosome, and we have analyzed the results together with published data from Moroccan populations. North African Y-chromosomal diversity is geographically structured and fits the pattern expected under an isolation-by-distance model. Autocorrelation analyses reveal an east-west cline of genetic variation that extends into the Middle East and is compatible with a hypothesis of demic expansion. This expansion must have involved relatively small numbers of Y chromosomes to account for the reduction in gene diversity towards the West that accompanied the frequency increase of Y haplogroup E3b2, but gene flow must have been maintained to explain the observed pattern of isolation-by-distance. Since the estimates of the times to the most recent common ancestor (TMRCAs) of the most common haplogroups are quite recent, we suggest that the North African pattern of Y-chromosomal variation is largely of Neolithic origin. Thus, we propose that the Neolithic transition in this part of the world was accompanied by demic diffusion of Afro-Asiatic-speaking pastoralists from the Middle East.     

The Cell Adhesion Molecule DdCAD-1 in Dictyostelium Is Targeted to the Cell Surface by a Nonclassical Transport Pathway Involving Contractile Vacuoles

DdCAD-1 is a 24-kD Ca²⁺-dependent cell– cell adhesion molecule that is expressed soon after the initiation of development in Dictyostelium cells. DdCAD-1 is present on the cell surface as well as in the cytosol. However, the deduced amino acid sequence of DdCAD-1 lacks a hydrophobic signal peptide or any predicted transmembrane domain, suggesting that it may be presented on the cell surface via a nonclassical transport mechanism. Here we report that DdCAD-1 is transported to the cell surface via contractile vacuoles, which are normally involved in osmoregulation. Immunofluorescence microscopy and subcellular fractionation revealed a preferential association of DdCAD-1 with contractile vacuoles. Proteolytic treatment of isolated contractile vacuoles degraded vacuole-associated calmodulin but not DdCAD-1, demonstrating that DdCAD-1 was present in the lumen. The use of hyperosmotic conditions that suppress contractile vacuole activity led to a dramatic decrease in DdCAD-1 accumulation on the cell surface and the absence of cell cohesiveness. Shifting cells back to a hypotonic condition after hypertonic treatments induced a rapid increase in DdCAD-1–positive contractile vacuoles, followed by the accumulation of DdCAD-1 on the cell membrane. 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, a specific inhibitor of vacuolar-type H⁺-ATPase and thus of the activity of contractile vacuoles, also inhibited the accumulation of DdCAD-1 on the cell surface. Furthermore, an in vitro reconstitution system was established, and isolated contractile vacuoles were shown to import soluble DdCAD-1 into their lumen in an ATP-stimulated manner. Taken together, these data provide the first evidence for a nonclassical protein transport mechanism that uses contractile vacuoles to target a soluble cytosolic protein to the cell surface.The cellular slime mold Dictyostelium discoideum transits from the solitary amoeboid state to an organized multicellular structure during development. This process is initiated in cells upon the depletion of nutrients, leading to the expression of many developmentally regulated genes and the chemotactic migration of cells in response to extracellular cAMP. Cells stream in concentric rings and/or spirals toward aggregation centers, giving rise to multicellular entities called pseudoplasmodia or slugs. The migrating slugs eventually culminate in the formation of fruiting bodies consisting of primarily spores and stalk cells (for review see Loomis, 1975).Multicellularity during development is maintained by the expression of cell–cell adhesion molecules, which fall into two broad categories based on their sensitivity to EDTA (for reviews see Gerisch, 1980; Siu et al., 1988; Siu, 1990; Fontana, 1995; Bozzaro and Ponte, 1995). There are two types of EDTA-sensitive cell adhesion sites. The EDTA/EGTA-sensitive cell adhesion sites, also known as contact sites B, are mediated by the Ca²⁺-dependent cell adhesion molecule gp24/DdCAD-1 (Knecht et al., 1987; Brar and Siu, 1993), while the EDTA-sensitive/EGTA- resistant sites are probably mediated by a Mg²⁺-dependent cell adhesion molecule (Fontana, 1993). The molecular nature of the latter sites is not yet known. Both types of adhesion sites are responsible for cell–cell interactions in the early stages of development. Coinciding with the aggregation stage is the rapid accumulation of the cell adhesion molecule gp80, which mediates the EDTA-resistant cell adhesion sites or contact sites A (Muller and Gerisch, 1978; Siu et al., 1985; Kamboj et al., 1988, 1989). In postaggregation stages, the EDTA-resistant adhesion sites are mediated by the membrane glycoprotein gp150 (Geltosky et al., 1979; Siu et al., 1983; Gao et al., 1992).DdCAD-1 is expressed by cells soon after the initiation of development (Knecht et al., 1987). Antibodies raised against gel-purified DdCAD-1 specifically inhibit the EDTA/EGTA-sensitive cell–cell adhesion sites and block development (Loomis, 1988). We have purified DdCAD-1 to homogeneity and demonstrated that labeled soluble DdCAD-1 binds to cells in an EDTA/EGTA-sensitive manner (Brar and Siu, 1993). Binding of DdCAD-1 to cells is prevented when cells are precoated with anti– DdCAD-1 antibodies, consistent with a homophilic mode of interaction. In addition, binding of DdCAD-1 to cells inhibits cell reassociation, indicating that it contains only a single cell binding site.Recent cloning of the DdCAD-1 cDNA predicts a protein of 23,924 daltons (Wong et al., 1996). The deduced amino acid sequence of DdCAD-1 shows significant sequence similarities with members of the cadherin family, and it contains a Ca²⁺-binding motif residing in the carboxy-terminal region. Indeed, Ca²⁺ overlay experiments have shown that DdCAD-1 is a Ca²⁺-binding protein with multiple binding sites (Brar and Siu, 1993; Wong et al., 1996). It is therefore conceivable that DdCAD-1 is a primitive member of the cadherin superfamily and it may mediate cell–cell adhesion in a manner similar to that of cadherins (Shapiro et al., 1995; Nagar et al., 1996). Another novel feature of the predicted sequence is that it lacks an amino-terminal hydrophobic signal peptide or a transmembrane domain, suggesting that DdCAD-1 is a soluble protein. Consistent with this observation, both subcellular fractionation and immunofluorescence microscopy have revealed a predominant cytoplasmic localization of DdCAD-1, indicating that 60–80% of DdCAD-1 is soluble (Brar and Siu, 1993; Sesaki and Siu, 1996). However, IgG binding and capping experiments clearly demonstrate that a substantial amount of DdCAD-1 is present on the cell surface (Brar and Siu, 1993; Wong et al., 1996). Interestingly, DdCAD-1 undergoes rapid translocation from the cytoplasm to the plasma membrane in the preaggregation stage of development (Sesaki and Siu, 1996), and then it becomes concentrated on filopodial structures and in cell– cell contact regions. These observations thus raise the question of how DdCAD-1 is transported and anchored to the cell surface.In this report we present morphological and biochemical evidence that DdCAD-1 is transported to the cell surface from the cytosol via contractile vacuoles, which is known so far to function exclusively in osmoregulation in cells. Furthermore, we show that isolated contractile vacuoles selectively take up soluble DdCAD-1 into their lumen in a cell-free system. Our results demonstrate, for the first time, a protein targeting function for contractile vacuoles and a novel nonclassical protein transport mechanism.     

Taxonomic revision and phytogeographic studies in Euphorbia (Euphorbiaceae) in the Khorassan provinces of Iran

This study is focused on the genus Euphorbia L. in a part of northeast Iran, viz. the three Khorassan provinces. Since there are many taxa of Euphorbia in Iran which are used in different industries and have significant effects on human and non‐human life it is important to revise their taxonomy. With about 90 species, following Turkey with 91 species, Iran is the second richest country for Euphorbia in Asia. Of these, 30 species are distributed in the Khorassan provinces. This is the first comprehensive work on the genus in this region. According to ‘Flora Iranica’, there are 17 species of Euphorbia in northeast Iran, while according to our results, there are 30 species of Euphorbia in the Khorassan provinces alone. In addition to various new taxonomic and biogeographic results, a new species, viz. E. chamanbidensis, is described. Euphorbia chamanbidensis is closely related to E. aucheri, but seed micro‐morphological characters differentiate them. Two identification keys to the Euphorbia species of the studied area are provided, one based on macro‐morphological characters and another based on seed micro‐morphological characters. Phytogeographic analysis and distribution maps for all species are also presented.     

A Probability Analysis of Historical Pregnancy and Fetal Data from Dutch Belted and New Zealand White Rabbit Strains from Embryo–Fetal Development Studies

Embryo‐fetal development (EFD) studies, typically in pregnant rats and rabbits, are conducted prior to enrolling females of reproductive age in clinical trials. Common rabbit strains used are the New Zealand White (NZW) and Dutch Belted (DB). As fetal abnormalities can occur in all groups, including controls, Historical Control Data (HCD) is compiled using data from control groups of EFD studies, and is used along with each study's concurrent control group to help determine whether fetal abnormalities are caused by the test article or are part of background incidences. A probability analysis was conducted on 2014 HCD collected at Charles River Inc., Horsham PA on Covance NZW, Covance DB, and Charles River (CR) NZW rabbits. The analysis was designed to determine the probability of 2 or 3 out of a group of 22 does aborting their litter or of having a fetal abnormality by chance. Results demonstrate that pregnancy parameters and fetal observations differ not only between strains, but between sources of rabbits of the same strain. As a result the probability of these observations occurring by chance in two or three litters was drastically different. Although no one single strain is perfect, this analysis highlights the need to appreciate the inherent differences in pregnancy and fetal abnormalities between strains, and points out that an apparent isolated increased incidence of an observation in one strain will not necessarily be test‐article related in another strain. A robust HCD is critical for interpretation of EFD rabbit studies, regardless of the rabbit strain used     

Subtype-Selective Small Molecule Inhibitors Reveal a Fundamental Role for Nav1.7 in Nociceptor Electrogenesis,Axonal Conduction and Presynaptic Release

Human genetic studies show that the voltage gated sodium channel 1.7 (Na_v1.7) is a key molecular determinant of pain sensation. However, defining the Na_v1.7 contribution to nociceptive signalling has been hampered by a lack of selective inhibitors. Here we report two potent and selective arylsulfonamide Na_v1.7 inhibitors; PF-05198007 and PF-05089771, which we have used to directly interrogate Na_v1.7’s role in nociceptor physiology. We report that Na_v1.7 is the predominant functional TTX-sensitive Na_v in mouse and human nociceptors and contributes to the initiation and the upstroke phase of the nociceptor action potential. Moreover, we confirm a role for Na_v1.7 in influencing synaptic transmission in the dorsal horn of the spinal cord as well as peripheral neuropeptide release in the skin. These findings demonstrate multiple contributions of Na_v1.7 to nociceptor signalling and shed new light on the relative functional contribution of this channel to peripheral and central noxious signal transmission.