Transforming growth factor-beta1-dependent activation of Smad2/3 and up-regulation of PAI-1 expression is negatively regulated by Src in SKOV-3 human ovarian cancer cells

The net balance between urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) has been implicated in tumor cell invasion and metastasis. To elucidate the mechanism of the transforming growth factor-beta1 (TGF-beta1)-dependent up-regulation of PAI-1 expression, we investigated which signaling pathway transduced by TGF-beta1 is responsible for this effect. Here, we show (1) nontoxic concentrations of TGF-beta1 up-regulates uPA expression in HRA and SKOV-3 human ovarian cancer cells, (2) TGF-beta1 activates Smads (phosphorylation of Smad2 and nuclear translocation of Smad3) and subsequently up-regulates PAI-1 expression in HRA cells, whereas TGF-beta1 neither activates Smads nor up-regulates PAI-1 in SKOV-3 cells, (3) pharmacological Src inhibitor PP2 or antisense (AS) c-Src oligodeoxynucleotide (ODN) treatment significantly induces TGF-beta1-dependent activation of Smads, leading to PAI-1 synthesis, compared with controls, in SKOV-3 cells, (4) combination of TGF-beta1 and PP2, which activates PAI-1 expression and reduces uPA expression in SKOV-3, results in decreased invasiveness, (5) pharmacological inhibitors for mitogen-activated protein kinase (MAPK) (PD98059) and phosphoinositide-3-kinase (PI3K) (LY294002 and wortmannin) or AS-PI3K ODN transfection do not affect TGF-beta1-induced Smad signaling and up-regulation of PAI-1 expression in SKOV-3 cells pretreated with PP2, and (6) the induction of PAI-1 protein was partially inhibited by an inhibitor of Sp1-DNA binding, mithramycin, implicating, at least in part, Sp1 in the regulation of this gene by TGF-beta1. In conclusion, TGF-beta1-dependent activation of Smad2/3, leading to PAI-1 synthesis, may be negatively regulated by Src, but not its downstream targets MAPK and PI3K in SKOV-3 cells. These data also reflect the complex biological effect of uPA-PAI-1 system.     

Activation of protein kinase C suppresses fragmentation of pig oocytes aged in vitro

When cultured for an extended time, pig oocytes that matured in vitro to the stage of metaphase II undergo the complex process designated as ageing. Under our conditions, some pig oocytes aged 3 days remained at the stage of metaphase II (22%), but others underwent spontaneous parthenogenetic activation (45%), and still others perished through fragmentation (28%) or lysis (5%). Activation of protein kinases C (PKCs) using phorbol-12-myristate-13-acetate (PMA) protects oocytes from fragmentation. None of the oocytes were fragmented after 3 days of aging in 50 nM of PMA. A similar effect (8% of fragmented oocytes) was observed after a 3-day treatment of aging oocytes with 100 μM of 1-stearoyl-2arachidonoyl-sn-glycerol (STEAR). PMA and STEAR activate both calcium-dependent and calcium-independent PKCs. This combined effect on PKCs seems to be essential for the protection of oocytes from fragmentation. Neither the specific activator of calcium-dependent PKCs 1-oleoyl-2-acetyl-sn-glycerol (OLE) nor the specific activator of calcium-independent PKCs dipalmitoyl-l-α-phosphatidylinositol-3,4,5-triphosphate heptaammonium salt (DIPALM) suppressed the fragmentation of aging pig oocytes. Twenty-one percentage of oocytes fragmented when aged for 3 days in 10 μM OLE and 26% of aged oocytes fragmented in 100 nM of DIPALM. However, fragmentation was significantly suppressed to 7% when the oocytes were exposed to the combination of both 10 μM OLE and 100 nM DIPALM. Aging pig oocytes cultured for 1 day with PMA maintained a high capability of being parthenogenetically activated (86% of activated oocytes), using calcium ionophore with 6-dimethylaminopurine. Ageing oocytes treated with PMA also had high capability of cleavage (82%) after their artificial parthenogenetic activation. However, their ability to develop to the stage of blastocyst (12%) was suppressed when compared with oocytes activated immediately after their maturation (29%).     

脑缺血/再灌对蒙古沙土鼠海马突触体酪氨酸磷酸化的影响

用蒙古沙土鼠双侧颈总动脉结扎（ＢＣＡＯ）前脑缺血模型,研究缺血／再灌对海马突触体蛋白酪氨酸磷酸休的影响及ＮＭＤＡ受体（ＮＲ）非竞争性拮抗剂氯胺酮（Ｋｅｔａｍｉｎｅ,ＫＴ）、Ｌ－型电压门控钙离子通道（Ｌ－ｔｙｐｅ ｖｏｌｔａｇｅ ｇａｔｅｄｃａｌｃｉｕｍ ｃｈａｎｎｅｌ,Ｌ－型ＶＧＣＣ）拮抗剂硝苯吡啶（ｎｉｆｅｄｉｐｉｎｅ,ＮＤ）及非ＮＲ拮抗６,７－二硝基喹恶啉上卫四（６,７－ｄｉ－ｎｉｔｒｏｐｕ     

Carboxy group derivatization for enhanced electron‐transfer dissociation mass spectrometric analysis of phosphopeptides

A novel strategy based on carboxy group derivatization is presented for specific characterization of phosphopeptides. By tagging the carboxy group with 1‐(2‐pyrimidyl) piperazine (PP), the ion charge states of phosphopeptides can be largely enhanced, showing great advantages for sequencing phosphorylated peptides with electron‐transfer dissociation MS. Besides, after PP‐derivatization, most non‐specific bindings can be avoided by eliminating the interaction between the carboxy group and TiO₂, greatly improving the specificity of TiO₂‐based phosphopeptide enrichment strategy. Moreover, being tagged with a hydrophobic group, the retention time of phosphopeptides in RPLC can be prolonged, overcoming the difficulty of separating phosphopeptides in RPLC‐based approach. Together with several other advantages, such as ease of handling, rapid reaction time, broad applicability and good reproducibility, this PP‐derivatization method is promising for high‐throughput phosphoproteome research.     

Early Palaeogene Louisinidae (Macroscelidea,Mammalia), their relationships and north European diversity

Seventeen species from the Palaeocene and Early Eocene of northern Europe, of which 12 are new, are described belonging to the extinct macroscelidean family Louisinidae, raised here from subfamily rank. These species belong to nine genera, of which five are new. The new genera are Walbeckodon , Berrulestes , Gigarton , Thryptodon , and Prolouisina . The new species are Walbeckodon krumbiegeli , Walbeckodon girardi , Paschatherium levei , Berrulestes phelizoni , Berrulestes pellouini , Berrulestes poirieri , Gigarton meyeri , Gigarton sigogneauae , Gigarton louisi , Thryptodon brailloni , Louisina marci , and Teilhardimys brisswalteri . Prolouisina is erected for ‘Louisina’atavella Russell, 1964. Cladistic analysis was undertaken to understand the relationships within the Louisinidae and between them and the North American family Apheliscidae, in which they had earlier been included as a subfamily. Louisinidae are shown to be sister group to a clade consisting of Apheliscidae plus Amphilemuridae and part of a paraphyletic and polyphyletic Adapisoricidae, all of which are tentatively considered to be stem members of the order Macroscelidea. The most primitive macroscelidid, Chambius, from the Early Eocene of northern Africa is nested within Apheliscidae when postcranial characters were included, but in a majority of cases within the Louisinidae when postcranial characters were excluded. Most species from northern Europe became extinct at the end of the Palaeocene, although the genus Paschatherium survived for much of the Early Eocene and Teilhardimys survived into the earliest Eocene. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 164 , 856–936.     

Variability in nitrogen stable isotope ratios of macroalgae: consequences for the identification of nitrogen sources

In our research, we collected and analyzed numerous macroalgal specimens (738) for isotopic analysis sampled over a year at monthly intervals across 20 sites within the Urías lagoon complex, a typical subtropical coastal ecosystem located in the Gulf of California. We quantified and characterized (chemically and isotopically) the N loads received by Urías throughout a year. We studied the spatial‐temporal variation of the chemical forms and isotopic signals of the available N in the water column, and we monitored in situ different environmental variables and other hydrodynamic parameters. Multiple N sources (e.g., atmospheric, sewage, seafood processing, agriculture and aquaculture effluents) and biogeochemical reactions related to the N cycle (e.g., ammonia volatilization, nitrification and denitrification) co‐occurring across the ecosystem, result in a mixture of chemical species and isotopic compositions of available N in the water column. Increased variability was observed in the δ¹⁵N values of macroalgae (0.41‰–22.67‰). Based on our results, the variation in δ¹⁵N was best explained by spatio‐temporal changes in available N and not necessarily related to the N sources. The variability was also explained by the differences in macroalgal biology among functional groups, species and/or individuals. Although the δ¹⁵N‐macroalgae technique was a useful tool to identify N sources, its application in coastal ecosystems receiving multiple N sources, with changing environmental conditions influencing biogeochemical processes, and high diversity of ephemeral macroalgal species, could be less sensitive and have less predictive power.