AbstractPinussibirica sparse growths. Based on clustering studied localities on species diversity similarity two groups of sites are defined: low-mountain sites and subalpine sparse growths of Pinussibirica ones.The distribution of millipedes along an altitudinal gradient in the south of Lake Teletskoye, Altai, Russia based on new samples from the Kyga Profile sites, as well as on partly published and freshly revised material , is esta Sibiriulusaltaicus Gulička, 1972 and specimens of the family Diplomaragnidae from the study localities have been made earlier : GPS (WGS84) position, altitude, habitat, sampling date, sampling methods.1: 51°20'47,3"N, 87°51'14,2"E, 443 m a.s.l., Pinussylvestris and Betulapendula forest with Larixsibirica, Abiessibirica and Pinussibirica, 12.08.2005, soil sampling.PageBreak2: 51°20'29,3"N, 87°51'40,0"E, 494 m a.s.l., Abiessibirica and Pinussibirica forest with ferns, 12.08.2005, soil sampling.4: 51°19'53,3"N, 87°51'78,0"E, 675 m a.s.l., Abiessibirica forest with Pinussibirica and Betulapendula, 18.08.2005, soil sampling.5: 51°19'28,5"N, 87°52'4,8"E, 853 m a.s.l., Abiessibirica forest with Pinussibirica, Populustremula and Betulapendula, 18.08.2005, soil sampling and hand sampling.6: 51°19'31,6"N, 87°52'16,1"E, 942 m a.s.l., Populustremula forest with Abiessibirica and Pinussibirica, 18.08.2005, soil sampling and hand sampling.7: 51°19'31,2"N, 87°52'21,1"E, 960 m a.s.l., Abiessibirica, Pinussibirica and Populustremula forest, 18.08.2005, soil sampling and hand sampling.8: 51°19'30,4"N, 87°52'50,0"E, 1100 m a.s.l., Pinussibirica forest with Abiessibirica, 17.08.2005, soil sampling.8a: 51°19'23,6"N, 87°53'2,1"E, 1191 m a.s.l., Pinussibirica forest with Abiessibirica, 17.08.2005, soil sampling and hand sampling.9: 51°19'07,5"N, 87°53'15,0"E, 1414 m a.s.l., Pinussibirica forest with Abiessibirica, 17.08.2005, soil sampling.10: 51°18'58,5"N, 87°53'33,3"E, 1468 m a.s.l., Pinussibirica forest with Abiessibirica, 17.08.2005, soil sampling.10a: 51°18'43,7"N, 87°54'23,7"E, 1699 m a.s.l., sparse growths of Pinussibirica with Betulapendula and Abiessibirica, 16.08.2005, soil sampling.11: 51°18'41,3"N, 87°55'34,7"E, 1736 m a.s.l., old fire-site, Betularotundifolia and Salixglauca bushes with Pinussibirica and Abiessibirica sparse growths, 16.08.2005, soil sampling.12: 51°18'27,8"N, 87°54'57,4"E, 1847 m a.s.l., old fire-site, Betularotundifolia and Salixglauca bushes with Pinussibirica and Abiessibirica sparse growths, 16.08.2005, soil sampling and hand sampling.13: 51°18'09,4"N, 87°55'43"E, 1861 m a.s.l., subalpine Pinussibirica forest, 14.08.2005, hand sampling.13a: 51°18'24,1"N, 87°55'06,9"E, 1877 m a.s.l., subalpine sparse growths of Pinussibirica, 16.08.2005, soil sampling and hand sampling.14: 51°18'23,0"N, 87°55'22,1"E, 1903 m a.s.l., subalpine sparse growths of Pinussibirica, 14.08.2005, soil sampling.15: 51°18'24,5"N, 87°55'31,0"E, 1962 m a.s.l., subalpine sparse growths of Pinussibirica with Abiessibirica, 14.08.2005, soil sampling and hand sampling under stones.16: 51°18'33,6"N, 87°55'32,9"E, 2028 m a.s.l., subgoltsy sparse growths of Pinussibirica with Betularotundifolia and Salixglauca bushes, 15.08.2005, soil sampling and hand sampling.18: 51°18'30,0"N, 87°56'10,7"E, 2194 m a.s.l., Betularotundifolia and Salixglauca mountain tundra with Dryas and lichens, 15.08.2005, soil sampling and hand sampling under stones.19: 51°18'30,5"N, 87°56'21,7"E, 2267 m a.s.l., summit of Malaya Koliushta Mt., Betularotundifolia and Salixglauca rocky mountain tundra with Dryas, Festuca and lichens, 15.08.2005, soil sampling and hand sampling under stones.PageBreakAlso we collected some material from two additional sampling sites :A: 51°20'16,8"N, 87°51'47,6"E, about 500 m a.s.l., Duschekiafruticosa forest on the bank of the river Bayas, 12.08.2005, hand sampling.B: 51°18'05,4"N, 87°55'48,3"E, about 1900 m a.s.l., Pinussibirica sparse growths, 14.08.2005, hand sampling.The cluster analysis was performed using Statistica 10 .The material treated herein has been deposited mainly in the collection of the Altai State University, Barnaul, Russia (ASU), and partly shared also with the collection of the Institute of Biology and Soil Science, Far Eastern Branch, Russian Academy of Sciences, Vladivostok, Russia (IBSS), as indicated in the text. The species names include the literature references concerning Asian Russia only.Taxon classificationAnimaliaJulidaJulidaeGulička, 1963Julusghilarovi Gulička, 1963: 521, 520: figs.Julusghilarovi – Julusghilarovighilarovi – 1 male, 7 females, 10 juv. (ASU), site 1; 1 male, 4 females, 3 juv. (ASU), site A; 1 female (IBSS), site 4; 5 males, 12 females, 4 juv. (ASU), site 5; 4 males, 6 females, 5 juv. (ASU), site 6; 1 female, 3 juv. (ASU), site 7; 3 juv. (ASU), site 8; 1 male, 1 female (IBSS), 3 males, 8 females, 11 juv. (ASU), site 19.This species appears to be widespread in the south of Siberia, Russia: Novosibirsk Area, Kemerovo Area, Altai Province, Republic of Altai, Republic of Khakassia, southern part of Krasnoyarsk Province. It is very likely it also occurs in the adjacent part of the Republic of Tyva.PageBreakmeadows, and montane moss-stony tundras. In the Kyga Biogeocenosis Profile the animal prefers forest litter in low- and mid-mountain chern taiga forests up to about 1200 m a.s.l., and also recorded in rocky mountain tundra on the summit of Malaya Koliushta Mt. at 2267 m a.s.l. It is very likely that the species is displaced from subalpine sparse growths of Pinussibirica by the congener of Julusinsolitus. The numbers range from 0.5 to 18 ind./m2.High ecological plasticity of this species allows it to inhabit different habitats like small-leaved, mixed and dark coniferous forests, herbaceous and alpine Taxon classificationAnimaliaJulidaJulidaeMikhaljova, 2009Julusghilarovibrachydactylusnon  – Julusinsolitus Mikhaljova, 2009b: 66–67, 64: figs.Julusinsolitus – 4 males, 5 females, 4 juv. (ASU), site 9; 4 males, 1 female (IBSS), 6 males, 9 females, 10 juv. (ASU), site 10; 2 males, 6 females, 17 juv. (ASU), site 10a; 7 males, 17 females, 25 juv. (ASU), site 11; 20 males, 9 females, 49 juv., 1 fragm. (ASU), site 12; 1 male, 1 female, 1 juv. (ASU), site 13; 2 males, 4 females, 7 juv. (ASU), site 13a; 3 males, 8 females, 10 juv. (ASU), site 14; 1 female, 2 juv. (ASU), site 15; 14 males, 15 females, 22 juv. (ASU), site B; 10 males, 12 females, 15 juv. (ASU), site 16; 2 females, 6 juv. (ASU), site 18.The species appears to be spread only in the south of Siberia, Russia: Republic of Altai.Pinussibirica to mountain tundra with Betularotundifolia and Salixglauca. The maximum abundance is about 98 ind./m2 registered in the old fire-site of sparse growths of Pinussibirica now occupied with a succession of dwarf trees of Betularotundifolia and Salixglauca. The above female specimens are the first records in this species.This species has been described by Taxon classificationAnimaliaJulidaJulidaeJulusamurensis Gerstfeldt, 1859: 271.Julusamurensis – Pacifiiulusimbricatus Mikhaljova, 1982: 211, 212: figs.PacifiiulusimbricatusPageBreak – Pacifiiulusamurensis – 1 male (IBSS), site 9; 3 males (ASU), site 16; 1 male (ASU), site 18.This species is characterized by disjunctive area. The first distribution area is in the south of Siberia  and the second one spreads in the Russian Far East  and North-Eastern China.2), collected from the mid-mountain dark coniferous forest, and also from subgoltsy sparse growths of Pinussibirica and mountain tundra with dwarf bushes of Betularotundifolia and Salixglauca, with the maximum altitude registered is about 2194 m a.s.l.This species is characterized by euryoky, dwelling in Siberia in herbaceous meadows, small-leaved, mixed and dark coniferous forests, and subalpine meadows and montane tundras, up to 2500 m a.s.l. . In the Taxon classificationAnimaliaJulidaJulidaeGulička, 1972Cylindroiulus (Sibiriulus) altaicusSibiriulusaltaicus – Isobatessibiricus Gulička, 1963: 522: figs.Isobates (Orinisobates) sibiricus – Orinisobatessibiricus – 1 male (ASU), site 1; 1 female (ASU), site 8a.The species appears to be quite widespread in the south of Siberia, Russia: Kemerovo Area, Republic of Khakassia, Altai Province, Republic of Altai, southern part of Krasnoyarsk Province, Republic of Tyva, Chita Area. Also it has been recorded in Eastern Kazakhstan and Kyrgyzstan.2.The species inhabits forest litter of small-leaved, mixed and dark coniferous forests, under bark of logs and trees, and in mosses and mushrooms. The maximum altitude registered is about 1700 m a.s.l. . In the Material examined. 1 juv. (ASU), site 2; 2 females, 1 juv. (ASU), site 9; 1 female, 2 juv. (ASU), site 10.Remarks. It is very likely these females and juveniles appear to belong to Sibiriulusaltaicus or Pacifiiulusamurensis.Taxon classificationAnimaliaChordeumatidaDiplomaragnidaeDiplomaragnabakurovi Shear, 1990: 22, 23: figs.Diplomaragnabakurovi – PageBreakAltajosomabakurovi – Craspedosomadeplanatum Stuxberg, 1876a: 34, figs.Craspedosomadeplanatum – Altajosomapinetorum Gulička, 1972: 37: figs.Altajosomapinetorum – Diplomaragnadeplanata – Diplomaragnapinetorum – Altajosomadeplanatum – 2 females (ASU), site 14. and the city of Mariinsk (Kemerovo Area).PageBreak, collected only from the mixed forest in the lowest study site at 443 m a.s.l.This species originally described from the Katunskii Mt. Range, Central Altai at 1600–2200 m a.s.l. . In the Taxon classificationAnimaliaChordeumatidaDiplomaragnidaeDiplomaragnakemerovo Shear, 1990: 21, 20: figs.Diplomaragnakemerovo – Altajosomakemerovo – 4 females (ASU), site 7; 1 juv. (ASU), site 8; 7 juv. (ASU), site 13a; 1 female, 3 juv. (ASU), site 16., mainly collected in low- and mid-mountain chern taiga at 853–1100 m PageBreaka.s.l., but also found in subalpine sparse growths of Pinussibirica and subgoltsy sparse growths of Pinussibirica with Betularotundifolia and Salixglauca bushes, with the maximum altitude registered is about 2028 m a.s.l.The species dwells in different forest habitats like small-leaved, mixed and dark coniferous forests. In the Kyga Biogeocenosis Profile it is very rare , site 16., mainly collected by hand sampling in subalpine sparse growths of Pinussibirica at 1847–2028 m a.s.l.This species inhabits dark coniferous taiga forests at 1350–1750 m a.s.l. and the subalpine belt (= goltsy) at 1750–2000 m a.s.l. . In the Material examined. 1 female (ASU), site A; 1 female, 1 juv. (ASU), site 5; 5 juv. (ASU), site 8a; 4 juv. (ASU), site 9; 1 female, 14 juv. (ASU), site 10; 5 juv. (ASU), site 10a; 1 female, 9 juv. (ASU), site 11; 1 female, 1 fragm. (ASU), site 13; 1 female, 1 juv. (ASU), site 18; 1 female, 1 juv. (ASU), site 19.Remarks. The above specimens appear to belong to some species of Altajosoma or Sheariateletskaya.Taxon classificationAnimaliaChordeumatidaAnthroleucosomatidaeGulička, 1972Ghilaroviakygae Gulička, 1972: 39, 40: figs.Ghilaroviakygae – PageBreak18 males, 16 females (ASU), site 1; 9 males, 6 females (ASU), site 2; 2 males, 1 female (ASU), site A; 3 males, 9 females, 2 juv. (ASU), site 4; 24 males, 18 females, 2 juv. (ASU), site 5; 15 males, 15 females, 2 juv. (ASU), site 6; 21 males, 15 females, 1 juv., 1 fragm. (ASU), site 7; 5 males, 3 females (ASU), site 8; 6 males, 6 females, 14 juv. (ASU), site 8a; 2 females, 1 juv. (ASU), site 9; 2 males (ASU), site 10a; 3 males, 3 females (ASU), site 11; 3 males, 1 female, 8 juv. (ASU), site 12; 3 males, 1 female (ASU), site 12; 6 males, 8 females (ASU), site 13; 2 males, 5 females (ASU), site 13a; 5 males, 16 females (ASU), site 14; 1 male, 1 female, 1 fragm. (ASU), site 15; 2 males, 8 females (ASU), site B; 1 male (ASU), site 16.The species is known to occur in the Republic of Altai, Siberia, Russia only.Pinussibirica up to subgoltsy, with the maximum altitude registered is about 2028 m a.s.l.This species prefers to live in mixed and dark coniferous forests, and also known from subalpine habitats like golsty, with the maximum altitude registered is about 2000 m a.s.l. . In the Altajellidae Mikhaljova & Golovatch, 2001)Altajellapallida Altajellapallida Gulička, 1972: 42, 43: figs.Altajellapallida – Kirkayakuspallidus – 1 male (ASU), site 8; 1 female (ASU), site 9.This species is an endemic in the south of Lake Teletskoye, Republic of Altai, Siberia, Russia.PageBreak2. The above material appears to belong to topotypes, and this is the first record of the female specimen in this species.The species lives in dark coniferous forests up to 1350 m a.s.l. . In the Taxon classificationAnimaliaChordeumatidaAltajellidaeGulička, 1972Teleckophoronmontanum Gulička, 1972: 41: figs.Teleckophoronmontanum – 3 males, 1 female, 4 juv. (ASU), site 8; 1 female, 3 juv. (ASU), site 8a.The area of this species appears to encompass the Republic of Altai and the southern part of the Krasnoyarsk Province, both Siberia, Russia.2.This species inhabits dark coniferous forests and montane tundras. The maximum altitude registered is about 1000 m a.s.l. . In the Taxon classificationAnimaliaPolydesmidaPolydesmidaePolydesmusclavatipes Stuxberg, 1876a: 34, figs.Polydesmusclavatipes – SchizoturaniusclavatipesPageBreak – 4 males, 2 females, 4 juv. (ASU), site 1; 2 males, 2 females, 3 juv. (ASU), site 2; 2 males, 1 female, 1 juv. (ASU), site 4; 4 males, 2 females, 1 juv. (ASU), site 5; 2 males, 1 females (ASU), site 6; 6 males, 2 females, 11 juv. (ASU), site 7; 1 male, 1 female (ASU), site 8a; 1 female (ASU), site 9; 7 juv. (ASU), site 14; 3 juv. (ASU), site B.This species appears to be quite widespread in the south of Siberia, Russia: Tomsk, Novosibirsk and Kemerovo areas, Altai Province, Republic of Altai, Republic of Khakassia, southern part of Krasnoyarsk Province.2, but also collected from subalpine sparse growths of Pinussibirica at about 1903 m a.s.l.Being highly euryoecic, the species populates various forest habitats , and also meadows and glades. In the Kyga Biogeocenosis Profile the species prefers low- and mid-mountain chern taiga forests, where its numbers range from 0.5 to 10 ind./mTaxon classificationAnimaliaPolydesmidaPolydesmidaePolydesmustabescens Stuxberg, 1876a: 35, figs.Polydesmustabescens – Turanodesmussalairicus Gulička, 1963: 523, 522: figs; Schizoturaniussalairicus – Schizoturaniustabescens – 3 juv. (ASU), site 2; 2 females, 2 juv., 1 fragm. (ASU), site 4; 4 juv. (ASU), site 5; 17 juv. (ASU), site 7; 2 females, 1 juv. (ASU), site 8a; 1 female (ASU), site 9; 2 females (ASU), site B.Being rather widespread, the species is known to occur in the south of Siberia, Russia: Tomsk, Novosibirsk and Kemerovo areas, Altai Province, Republic of Altai, Republic of Khakassia, southern part of Krasnoyarsk Province.2, but also collected from subalpine sparse growths of Pinussibirica at about 1900 m a.s.l.This species lives in different forests like small-leaved, mixed and dark coniferous ones, and also populates meadows. In the Kyga Biogeocenosis Profile the animal prefers low- and mid-mountain chern taiga forests, where its numbers range from 0.5 to 8 ind./mPageBreakJulusghilarovighilarovi Gulička, 1963, Julusinsolitus Mikhaljova, 2009, Orinisobatessibiricus , Pacifiiulusamurensis , Sibiriulusaltaicus , Ghilaroviakygae Gulička, 1972, Kirkayakuspallidus  , Teleckophoronmontanum Gulička, 1972, Altajosomabakurovibakurovi , Altajosomadeplanatum , Altajosomakatunicum Mikhaljova, 2000, Altajosomakemerovo , Sheariateletskaya Mikhaljova, 2000, Schizoturaniusclavatipes  and Schizoturaniustabescens .The millipede diversity in the south of Lake Teletskoye is estimated to be at least 15 species and subspecies from 10 genera, 6 families and three orders: Betularotundifolia and Salixglauca, achieving from 5 to 9 species, whereas subalpine sparse growths of Pinussibirica are characterized by the lowest millipede diversity, with 3 the most widespread species , evidently caused by the abundance of plant debris of dwarf vegetation of Betularotundifolia and Salixglauca that appear to be more suitable for feeding of millipedes than pine litter.The numbers of diplopods range from 14.5 to 65 ind./m² in subzones of low- and mid-mountain chern taiga forests, and from 5.5 to 8 ind./m² in high-mountain shrub tundras , and also the highest locality of chern taiga forest (site 10) and a transition locality from subalpine sparse growths to shrub mountain tundra (site 16), with altitudes range from 1468 to 2028 m a.s.l. Some localities of mid-mountain chern taiga forests and mountain tundras are less similar both to each other and to other groups.Clustering of investigated sites in the Kyga Biogeocenosis Profile on species diversity allows to grouping at least two obvious sets of sites Figure . The firJulus as regards to altitude distribution, showing competitive relationships to each other. Thus, Julusghilarovighilarovi is mainly recorded in low-mountain and in the beginning of mid-mountain chern taiga forests, while it almost disappears in subalpine sparse growths of Pinussibirica, but it emerges again in rocky mountain tundra on the top of the investigated biogeocenosis profile. At the same time, Julusinsolitus populates mainly subalpine sparse growths of Pinussibirica and lifts up to shrub mountain tundra at 2194 m a.s.l. Taking into account that Julusinsolitus is very abundant here, we assume it wins the competition and PageBreakPageBreakdisplaces Julusghilarovighilarovi from subalpine Pinussibirica sparse growths. The record of female specimens of Julusinsolitus is the first one in this species.The very interesting situation is observed with two congeners of Kirkayakidae, Kirkayakuspallidus and Teleckophoronmontanum, are reported in chern mid-mountain taiga at the highest altitudes for the first time. Also this is the first record of the female specimen in the former species.Both members of


Ptprz-deficient mice than in those of wild-type mice. We then investigated the role of Tyr-554 phosphorylation in cell motility control using three different methods: random cell motility, wound healing, and Boyden chamber assays. The shRNA-mediated knockdown of endogenous Git1 impaired cell motility in A7r5 smooth muscle cells. The motility defect was rescued by the exogenous expression of wild-type Git1 and a Git1 mutant, which only retained Tyr-554 among the multiple potential tyrosine phosphorylation sites, but not by the Tyr-554 phosphorylation-defective or phosphorylation-state mimic Git1 mutant. Our results suggested that cyclic phosphorylation-dephosphorylation at Tyr-554 of Git1 was crucial for dynamic interactions between Git1 and paxillin/Hic-5 in order to ensure coordinated cell motility.G protein-coupled receptor kinase-interactor 1 (Git1) is involved in cell motility control by serving as an adaptor that links signaling proteins such as Pix and PAK to focal adhesion proteins. We previously demonstrated that Git1 was a multiply tyrosine-phosphorylated protein, its primary phosphorylation site was Tyr-554 in the vicinity of the focal adhesion targeting-homology (FAH) domain, and this site was selectively dephosphorylated by protein tyrosine phosphatase receptor type Z (Ptprz). In the present study, we showed that Tyr-554 phosphorylation reduced the association of Git1 with the FAH-domain-binding proteins, paxillin and Hic-5, based on immunoprecipitation experiments using the Tyr-554 mutants of Git1. The Tyr-554 phosphorylation of Git1 was higher, and its binding to paxillin was consistently lower in the brains of  PAKs) interacting exchange factor (Pix) family of Rho guanine nucleotide exchange factors [www.phosphosite.org). Although Git1 is tyrosine-phosphorylated in a Src-dependent manner [Git1, which is also referred to as Cool-associated, tyrosine-phosphorylated 1 (Cat-1), is a member of the GTPase-activating protein for ADP-ribosylation factor (Arf GAP) family , 3, anky factors , 7–10. G factors , 6, 8, w factors , 8. Git1 factors , and 13 t manner , 13, thet manner . Git1 wat manner .glycosylphosphatidylinositol (GPI)-anchored molecule, F3 [Reversible protein tyrosine phosphorylation controlled by protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs) is critical for the regulation of numerous cellular events including cell adhesion and migration . PTP reccule, F3 .membrane-associated guanylate kinase, WW and PDZ domain-containing 1 (Magi1) at Tyr-373, and paxillin at Tyr-118 [As substrate molecules that transduce signals downstream of Ptprz, we previously identified several cellular proteins including Git1 by developing a genetic method named “the yeast substrate-trapping system” to screen for PTP substrates , 21. The Tyr-118 . We cons Tyr-118 .Ptprz-deficient mice, in which a maturation-dependent enhancement in long-term potentiation in the CA1 region of the hippocampus along with learning deficits [Ptprz-deficient mice [Git1-knockout mice were previously reported to exhibit deficits in the acquisition of conditioned fear [Ptprz-deficient mice were shown to be resistant to gastric ulceration by VacA, a cytotoxin secreted by Helicobacter pylori [The physiological importance of Ptprz has been demonstrated through studies using deficits –24, altedeficits , 26, anddeficits  were repent mice . Git1 isent mice , 29, andned fear  and impaned fear . In perined fear . Ptprz-dr pylori . The tyrr pylori .Among the multiple phosphorylation sites in Git1 by Src, Ptprz preferentially dephosphorylated phospho-Tyr-554 . We assuThe following are the specificities and sources of antibodies used: Against phosphotyrosine , the FLAG epitope , the Myc epitope , GFP , Hic-5 , paxillin , and Git1 . Rabbit antisera specific for the amino acid residues 251–555 of Git1 (anti-GIT1/Cat-1) , and a rThe plasmid series of pFLAG-Git1 were used for the expression of FLAG-tagged Git1 and its tyrosine mutants . The pla2. The DNA transfection of HEK293T cells was performed using the standard calcium phosphate technique [Git1 in A7r5 cells was performed with the vector pLKO.1-Git1, cells were selected with puromycin (5 μg/ml).HEK293T cells  were maintained on dishes coated with rat tail collagen in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a humidified incubator at 37°C with 5% COechnique . A7r5 (rg for 15 min. The supernatants were mixed with anti-FLAG M2 magnetic beads (Sigma) by rotation for 3 h. After washing the beads, the bound proteins were eluted with FLAG elution solution (Sigma) according to the manufacturer’s instructions. In the co-immunoprecipitaion assays, the bound proteins were eluted by boiling with SDS-PAGE sample buffer and subjected to Western blotting.In order to screen the molecules that bind to Git1, HEK293T cells transfected with the indicated plasmids were treated with 100 μM pervanadate for 15 min. Cells were extracted with lysis buffer, 20 mM Tris-HCl, pH 7.4, 1% NP-40, 150 mM NaCl, 10 mM NaF, 1 mM sodium orthovanadate, and an EDTA-free protease inhibitor cocktail , and then centrifuged at 15,000 in vivo, 4-month-old Ptprz-deficient [To analyze tyrosine phosphorylation eficient  and wildeficient . The synSamples were subjected to SDS-PAGE followed by semi-dry electroblotting onto a polyvinylidene difluoride (PVDF) membrane . The membrane was incubated for 1 h in a blocking solution , and incubated overnight with the respective antibodies in the blocking solution. The binding of these antibodies was detected with an ECL Western blotting system . In order to detect phosphorylated proteins, the membranes were blocked with 1% BSA and 0.1% Triton X-100 in 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and then incubated with the HRP-conjugated anti-phospho-Tyr mAb (clone PY20) or rabbit anti-pY554-Git1 antibodies.2-controlled chamber . The expression of mCherry-Git1 was verified using an appropriate filter set  before initiating image capturing. Phase contrast (PhC) images were taken with a 40x objective lens every 5 min for 3 h (for Wind-Rose plots), or every 10 sec for 30 min (for kymographs) per experiment. The paths of cell movement were traced by following nuclei between frames of the movies, and the trajectory length from the traced images was measured with Adobe Photoshop CS6 (Adobe). Kymographs were generated using ImageJ (rsb.info.nih.gov) installed with multiple kymograph plugins. The resulting image represented membrane activity (y axis) over time (x axis). The protrusion frequency was defined as the number of peaks extending >0.5 μm.A7r5 cells and its transfectants were replated on fibronectin (50 μg/ml)-coated glass-bottomed dishes in OPTI-MEM (Life Technologies) supplemented with 2% FBS. Live-cell imaging was performed using a microscope system equipped with a temperature- and COGit1-knockdown cells transfected with mCherry or mCherry-fused Git1 constructs were cultured for 48 h, and mCherry-positive cells were sorted by flow cytometry . Boyden chamber assays were performed using 24-well transwell inserts with an 8-μm pore-sized membrane  and 24-well culture plates . Cells in 250 μl of OPTI-MEM containing 100 μg/ml BSA were added to a fibronectin (50 μg/ml)-coated transwell insert (1.5 × 105 cells per well). The lower chambers were filled with 200 μl of the medium containing epidermal growth factor  as a chemoattractant. After a 6-h incubation, cells fixed with 10% neutral formalin were stained with DAPI . Cells present in the upper chamber were removed with cotton swabs, and cells that migrated to the lower surface were counted using a conventional fluorescence microscope. In the wound healing assay, cells (1.0 × 105 cells per well) were replated on fibronectin (50 μg/ml)-coated 24-well plastic plates , and then cultured with DMEM supplemented with 10% FBS until monolayers formed. After changing the medium to OPTI-MEM containing 100 μg/ml BSA and 10 ng/ml EGF, cell monolayers were scratched with a sterile micropipette tip, and then allowed to migrate for 48 h.Parental A7r5 and Git1 was preferentially phosphorylated at Tyr-554, which was located between the SHD and FAH domains see . To examHic-5 and its family member, paxillin, contain LD (leucine-aspartate repeat) motifs at the N-terminus and LIM  domains at the C-terminus . Previou9F-Y554 showed decreased binding to Hic-5 with the pervanadate treatment as well as WT (lanes 4 and 9): Y9F-Y554 is only phosphorylated at Tyr-554; see also [To determine the effects of Tyr-554 phosphorylation on the binding of Git1 to Hic-5 and paxillin, we exogenously expressed FLAG-tagged wild-type or mutant Git1 in HEK293T cells, together with Myc-tagged Hic-5. We verified the appropriate protein expression of transfected molecules and intensive tyrosine phosphorylation of cellular proteins by the pervanadate treatment . The robsee also . Thus, tAs shown in Ptprz-deficient mice [Ptprz-deficient mice than in wild-type mice  of Git1 that forms a coiled-coil structure and mediates homodimerization , 10, we Git1 mRNA. Western blotting showed that the amount of endogenous Git1 was less than 20% in knockdown (Git1-KD) cells as compared with that in parental cells   , indicat554D did . Similar554D did  and cell554D did . These rGit1-KD cells was defective in contrast to parental cells  of Git1, and the FAH-deleted Git1 mutant no longer binds to paxillin . The phoThe binding capacity of the phosphorylation-state mimic Y554D to Hic-5 and paxillin was further reduced by pervanadate Figs.  and 3. TGit1 resulted in impairments in cell migration activity (Figs. Git1-KD cells, in contrast to wild-type or Y9F-Y554 (Figs. 9F-Y554, but also Y554D rescued lamellipodial protrusion activity in Git1-KD cells, whereas Y554F did not (The knockdown of ty Figs.  and 8 asty Figs.  in A7r5  did not . TherefoThese results are reminiscent of the finding that Git1 has a similar mechanism to that observed in vinculin, one of the core focal adhesion proteins . VinculiIn conclusion, we herein revealed the functional significance of Tyr-554 phosphorylation in Git1, the primary phosphorylation site by Src. The specific dephosphorylation at Tyr-554 by Ptprz appears to fine tune the targeting of Git1-Pix complexes to appropriate subcellular locations . We cons


Chronic lymphocytic leukemia (CLL), the most common adult leukemia, remains incurable despite advancements in treatment regimens over the past decade. Several expression profile studies have been pursued to better understand CLL pathogenesis. However, these large-scale studies only provide information at the transcriptional level. To better comprehend the differential protein changes that take place in CLL, we performed a reverse-phase protein array (RPPA) analysis using 167 different antibodies on B-cell lysates from 18 CLL patients and 6 normal donors. From our analysis, we discovered an enrichment of protein alterations involved with mRNA translation, specifically upregulation of the translation initiator eIF4G and phosphorylation of the cap-dependent translation inhibitor 4E-BP1 at serine 65. Interestingly, 4E-BP1 phosphorylation occurred independently of AKT phosphorylation, suggesting a disconnect between PI3K/AKT pathway activation and 4E-BP1 phosphorylation. Based on these results, we treated primary CLL samples with NVP-BEZ235, a PI3K/mTOR dual inhibitor, and compared its apoptotic-inducing potential against the BTK inhibitor Ibrutinib and the PI3Kδ inhibitor Idelalisib. We demonstrated that treatment with NVP-BEZ235 caused greater apoptosis, greater apoptotic cleavage of eIF4G, and greater dephosphorylation of 4E-BP1 in primary CLL cells. Taken together, these results highlight the potential dependence of eIF4G overexpression and 4E-BP1 phosphorylation in CLL survival. SF3B1, NOTCH1, and MYD88 [ZAP70, LPL, CRY1, and LDOC1 [CD38 expression status and have provided better comprehension of the molecular abnormalities that occur in this disease [Chronic lymphocytic leukemia (CLL) is a malignant disease characterized by the accumulation of monoclonal B cells that have escaped their regulated life cycle. CLL is the most common adult leukemia in the Western world, and though it allows longer survival than most leukemias, this disease creates a poor quality of life and currently remains incurable . Particund MYD88 -6, as wend LDOC1 . Along w disease , 15-17. Understanding how pro-proliferative proteins are differentially altered in CLL has currently gained popularity based on recent studies that demonstrate the efficacy of targeting signaling proteins involved in the B-cell Receptor (BCR) pathway -22. Mostex vivo treatment results with the dual PI3K/mTOR inhibitor NVP-BEZ235 help demonstrate the potential dependence of mRNA translation in CLL survival, as well as revealing mRNA translation as a potential therapeutic target in CLL.Though several proteins have individually been investigated when determining the oncogenic features of CLL, no current study has simultaneously examined numerous protein alterations on a comprehensive level. Based on this current void, our goal was to perform reverse-phase protein array (RPPA) analysis using 167 antibody probes on primary CLL lysates in order to determine the deregulated protein events that occur in CLL. From this study, we demonstrate that the AKT/mTOR-related proteins are altered in CLL, with significant alteration occurring in the downstream mRNA translational machinery proteins eukaryotic translation factor 4G (eIF4G) and the eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). These results and corresponding CD38 expression, FISH, ZAP70 expression, treatment, and Rai stage status in order to determine whether these clinical parameters demonstrate any large-scale protein signatures  with 38 probes being upregulated and 20 being downregulated in CLL  and AKT-dependent mTOR S2448 phosphorylation when comparing the three treatments . HoweverInterestingly, this decrease in 4E-BP1 phosphorylation also correlated with greater cleavage of the eIF4G protein. This event previously characterized in immortalized lymphoma cell lines treated with chemotherapy , currentBy conducting RPPA analysis on primary CLL samples using 167 different antibody probes, we have been able to provide a comprehensive view of specific protein signaling and expression changes that take place in CLL. Specifically, we were able to determine from our selected samples that CLL contains a common protein expression signature that is seemingly more consistent among the CLL subtypes characterized by different genetic abnormalities. This common protein signature coincides with previous results demonstrating general overexpression of BCR-related signaling proteins, like BTK, regardless of a patient's clinical subtype , 23, 24.By further analyzing our clinical RPPA dataset through differential expression analysis between CLL and healthy B-cells, we revealed upregulation of proteins that are commonly associated with CLL pathogenesis, including apoptotic resistance  and PI3K/AKT/mTOR pathway proteins  , 36, 39,Understanding the expression and activation states of numerous proteins simultaneously allows for better knowledge of how to target these oncogenic events in CLL. Within the realm of the AKT/mTOR pathway, we specifically determined that proteins involved in mRNA translational regulation were being upregulated in CLL patient samples, revealing a potential characteristic of CLL oncogenesis. These aberrant events regarding protein translation include overexpression of the translation initiator eIF4G and phosphorylation of the cap-dependent translation regulator 4E-BP1, an event directly regulated by mTOR activity . These tNevertheless, the intriguing aspect of this translational machinery upregulation was its uncoupling from BCR-mediated AKT activation. Though total AKT and mTOR are upregulated in our RPPA dataset, the PI3K/AKT-related phosphorylation activity for these proteins appears to be negligible. This type of result is expected as the RPPA CLL patient samples were collected from the peripheral blood and are not in their active state, which occurs in the lymph node. However, 4E-BP1 serine 65 phosphorylation, an event that typically is regulated by the cascading phosphorylation of the PI3K/AKT/mTOR pathway , was hypCurrently, we do not know the specific mechanisms that mediate sustained 4E-BP1 phosphorylation in the absence of AKT activation. One possibility could be the dysregulation of phosphatases that mediate dephosphorylation of 4E-BP1. One particular phosphatase, PPM1G, has previously been identified as a 4E-BP1-specific phosphatase . HoweverThis potential disconnect between AKT activation and 4E-BP1 activation provided rationale for investigating the effectiveness of simultaneously targeting both upstream and downstream of the PI3K/AKT/mTOR pathway in CLL. We examined this concept by determining the inhibitory capability of the dual PI3K/mTOR inhibitor NVP-BEZ235. From our results, we saw that 4E-BP1 and eIF4G were significantly altered in CLL cells treated with NVP-BEZ235 when compared to Ibrutinib and Idelalisib, two drugs that target upstream BCR-related effectors involved in PI3K/AKT activation , 21. TheThus, with the coordinating results of our RPPA and inhibitor treatment comparisons in CLL patient samples, we demonstrate a rationale for considering mTOR-mediated mRNA translation as a therapeutic target in CLL. This general idea of targeting the mTOR pathway with rapamycin-based inhibitors has previously been investigated in CLL. However, current understanding of the rapamycin-induced negative feedback loop, which causes increased AKT signaling, provides explanation of why treating with single-agent rapalogs may have been ineffective in the clinical setting , 82. MorTM B-cell isolation kit . The isolated B-cells were then prepared in RIPA buffer supplemented with proteasome inhibitor and phosphatase inhibitor . The lysates were prepared to provide 1-1.5mg/ml of total protein lysate. RPPA analysis of the prepared lysates was then performed as previously described [Peripheral blood samples from CLL patients were obtained with patient consent and approved for use by the Institutional Review Board of Georgia Regents University. Peripheral blood samples from normal donors were purchased from a local blood bank. Once blood samples were obtained, CD19+ B-cells were isolated using the RosetteSepescribed . For thi7 cells per well of a 6-well dish and co-cultured with either Huh7.5 cells or HS-5 cells at a density of 1×106 cells per well of a 6-well dish. NVP-BEZ235, Ibrutinib (PCI-32765), and Idelalisib (GS-1101) were obtained from Selleck Biochem . Inhibitors were reconstituted in DMSO in order to treat primary CLL cells at a 10uM concentration.All cell culture experiments were maintained in RPM1640 Medium with 1% Penn/Strep  and 10% Human Serum . For co-culture experiments, primary CLL cells were seeded at 1×10Cell proliferation of the MEC1 CLL cell line was measured using the PrestoBlue Cell Viability reagent . Briefly, 25,000 MEC1 cells were treated with Ibrutinib, Idelalisib, or NVP-BEZ235 for 72 hours with a log-fold increase in concentration for each inhibitor. After 72 hours, MEC1 cells were incubated with PrestoBlue reagent for 2 hours and measured on a Spectramax Plus fluorescence plate reader  in order to determine an IC50 for each inhibitor in MEC1.Cell viability and apoptosis of inhibitor-treated primary CLL cells was assessed by Annexin V+/DAPI apoptotic assay using an Annexin V-FITC antibody  and DAPI staining . Annexin V and DAPI staining of primary cells were measured using an LSRII flow cytometer .Hierarchical clustering analysis for RPPA was performed using Partek Genomics Suite 6.6  and pathway analysis was performed using Ingenuity Pathway Analysis . Statistical measures for individual protein analysis and apoptotic studies were assessed using GraphPad Prism 6 .


Hyalomma marginatum rufipes, a vector of Crimean Congo hemorrhagic fever show weak similarity to the madanins, a group of thrombin-inhibitory peptides from a second tick species, Haemaphysalis longicornis. We have evaluated the anti-serine protease activity of one of these H. marginatum peptides that has been given the name hyalomin-1. Hyalomin-1 was found to be a selective inhibitor of thrombin, blocking coagulation of plasma and inhibiting S2238 hydrolysis in a competitive manner with an inhibition constant (Ki) of 12 nM at an ionic strength of 150 mM. It also blocks the thrombin-mediated activation of coagulation factor XI, thrombin-mediated platelet aggregation, and the activation of coagulation factor V by thrombin. Hyalomin-1 is cleaved at a canonical thrombin cleavage site but the cleaved products do not inhibit coagulation. However, the C-terminal cleavage product showed non-competitive inhibition of S2238 hydrolysis. A peptide combining the N-terminal parts of the molecule with the cleavage region did not interact strongly with thrombin, but a 24-residue fragment containing the cleavage region and the C-terminal fragment inhibited the enzyme in a competitive manner and also inhibited coagulation of plasma. These results suggest that the peptide acts by binding to the active site as well as exosite I or the autolysis loop of thrombin. Injection of 2.5 mg/kg of hyalomin-1 increased arterial occlusion time in a mouse model of thrombosis, suggesting this peptide could be a candidate for clinical use as an antithrombotic.A group of peptides from the salivary gland of the tick  The cleft is formed in part by the hydrophobic 60s-loop and the autolysis loop (centered approximately at residue 149) that act to limit access by potential substrates, creating a highly specific protease . Ch. ChHyalo-termini  23]. Th. ThHyalo-termini .Hyalomin-1 was synthesized without its putative signal sequence and tested for inhibition of eight coagulation proteases along with eight additional important serine proteases having functions not related to coagulation . In eachIn addition to the cleavage of fibrinogen, thrombin catalyzes a number of other important proteolytic reactions related to hemostasis. We tested the ability of hyalomin-1 to inhibit thrombin-mediated activation of additional macromolecular substrates involved in coagulation, platelet activation and fibrinolysis. The protease activated receptors PAR-1 and PAR-4 on platelets are cleaved by thrombin leading to activation and subsequent aggregation . Inhibit50 value of approximately 25 nM  of 11.9 ± 1.8 nM and 0.4 ± 0.1 nM at salt concentrations of 150 and 50 mM, respectively, as compared to reported values of 56 nM for madanin-1 and 32 nM for madanin-2 at an ionic strength of 50 mM (14). The linear progress curves obtained immediately after thrombin addition indicated rapid equilibrium binding of hyalomin-1, making it a “fast binding” inhibitor (a) for thrombin binding of 1 x 106 M-1s-1 and a dissociation rate constant (kd) of 0.02 s-1. The calculated dissociation equilibrium constant (KD) of 19 nM was consistent with the Ki obtained from steady-state kinetic results.Kinetic analyses showed hyalomin-1 to be a competitive inhibitor of S2238 hydrolysis by thrombin  with inhnhibitor . Bindingnhibitor . Analysi41-Leu motif is the most highly conserved region in the amino acid alignment from the three tick species shown in 54-Leu55 peptide bond contained within this motif [21, a residue that is not conserved in the hyalomins. Additionally, the madanins are cleaved by factor Xa at Arg16, Arg25 and Arg54. The former two positions are not conserved in the hyalomins, while the latter is equivalent to Arg41, the thrombin cleavage site [41-Leu42 peptide bond is the sole cleavage site. No full-length peptide (m/z 6381.3) remained at the end of the incubation period, demonstrating that cleavage proceeded to completion at a 25:1 peptide to enzyme ratio  as well the C-terminal portion of the mature peptide were also evaluated  manner at the catalytic site with Arg54 occupying the P1 position [in vivo, but testing of a sulfated variant of the 13–44 peptide suggests that this does not dramatically increase potency. Apparently,the N-terminal regions do not constitute an independent exosite-binding domain.A previously described crystal structure of the madanin-1-thrombin complex shows a four-residue madanin peptide sequence Alaposition . This biS1 ARRIVE Checklist(PDF)Click here for additional data file.


Labisia pumila (Kacip Fatimah) is a popular herb in Malaysia that has been traditionally used in a number of women’s health applications such as to improve libido, relieve postmenopausal symptoms, and to facilitate or hasten delivery in childbirth. In addition, the constituents of this plant have been reported to possess anticancer, antioxidant, and anti-inflammatory properties. Clinical studies have indicated that cytochrome P450s (CYPs), P-glycoprotein (P-gp), and Pregnane X receptor (PXR) are the three main modulators of drug-drug interactions which alter the absorption, distribution, and metabolism of drugs. Given the widespread use of Kacip Fatimah in dietary supplements, the current study focuses on determining the potential of its constituents to affect the activities of CYPs, P-gp, or PXR using in vitro assays which may provide useful information toward the risk of herb-drug interaction with concomitantly used drugs. Six compounds isolated from the roots of L. pumila  were tested, in addition to the methanolic extract. The extract of L. pumila showed a significant time dependent inhibition (TDI) of CYP3A4, reversible inhibition of CYP2C9 and 2C19 and a weak inhibition of 1A2 and 2D6 as well as an inhibition of P-gp and rifampicin-induced PXR activation. The alkyl phenols inhibited CYP3A4 (TDI), CYP2C9, and 2C19 (reversible) while saponins inhibited P-gp and PXR. In conclusion, L. pumila and its constituents showed significant modulation of all three regulatory proteins  suggesting a potential to alter the pharmacokinetic and pharmacodynamic properties of conventional drugs if used concomitantly. Labisia pumila (Blume) Fern.-Vill., locally termed as Kacip Fatimah (KF), is a popular herb in South East Asian countries. It belongs to the Myrsinaceae family. Recently it has been identified as one of the top five herbs used in Malaysia for treating variety of ailments  and MDCK-II cells by using two widely used substrates calcein-AM and digoxin. Modulation of PXR activity was monitored through a reporter gene assay in HepG2 cells transfected with PXR plasmid DNA and a luciferase reporter plasmid PCR5. Additionally, we used FDA guided assumptions , P-gp, and PXR using umptions  to predi3H-digoxin, 0.25 mCi/0.25 ml] was from Perkin Elmer Life Sciences . Troleandomycin was from Santa Cruz Biotechnology, Inc. . Preparation of L. pumila methanolic extract and isolation of its constituents used in the current study were described in our previous study  and hMDR1-MDCK-II (transfected) cell lines were a gift from Dr. Gottesman . Dulbecco’s Modified Eagle Medium (DMEM), Minimal Essential Medium (MEM), Hanks balanced salt solution (HBSS), HEPES, Trypsin EDTA, Penicillin-streptomycin, and Sodium Pyruvate were from GIBCO BRL . Fetal bovine serum (FBS) was from Hyclone Lab Inc. . CYP1A2/CEC, CYP2C9/MFC, CYP3A4/BQ, CYP2C19/CEC, and CYP2D6/AMMC high throughput inhibitor screening kits were from BD Gentest . Transwell plates  were from Costar Corp. . All other chemicals were from Sigma Chem. Co., . Radio labeled digoxin -resorcinol was effective in inhibiting CYP3A4 and 2C9 with IC50 values of 4.1 ± 0.2 and 11 ± 0.8 μM, respectively, while belamcandol B inhibited CYP2C19 with an IC50 value of 2.2 ± 0.6 μM.The methanolic extract and alkyl phenolic compounds showed dose dependent inhibition of CYP3A4, 2C9, and 2C19  were similar to the published literature values -pentadecenyl]-resorcinol, fatimahol, and methanol extract was 13.6, 5.6, and 4.7, respectively, .Based on these results, we further tested the TDI potential of extract and selected constituents toward CYP3A4, 2C9, and 2C19. The test samples were pre-incubated for 30 min with the co-factors, control protein, and specific enzymes before substrates were added. ICe values . In conts report . FurtherL. pumila and the constituents by using the two widely used probes calcein-AM and digoxin  but significantly less potent than the effect of cyclosporin A (EC50 8 ± 1.2 μM). The Papp value of LY was in the range of 1.1 ± 0.8 × 10-6 cm/s, which was similar to our previously published values  caused a fourfold induction in PXR activity which is in agreement with previous reports . On the other hand, the methanolic extract (3–30 μg/mL) and the two saponins  dose dependently decreased rifampicin-induced PXR activity . These results indicate that L. pumila and its constituents significantly modulate the activity of PXR and thereby could affect the downstream genes involved in PXR signaling. Additionally, no cytotoxicity was observed toward HepG2 cells with either KF methanolic extract or its constituents up to the highest tested concentration of 30 μg/mL or 30 μM (data not shown) confirming that the inhibition of PXR as seen with methanolic extract and the two saponins is not due to the toxicity toward HepG2 cells.Finally, we looked at the modulation of PXR activity by the extract and the constituents using a reporter gene assay in HepG2 cells. One of the alky phenols, fatimahol, significantly induced PXR activity (1.8-fold) at the highest tested concentration of 30 μM, while at lower concentration the effect was not significant Table 2. Since the intestinal absorption or plasma concentrations are not known for the test compounds, we made an assumption that all of the compounds are completely absorbed (100% bioavailable) from the GI tract. Based on this, the predicted GI and plasma concentrations of the compounds were calculated as shown in Tables 3. The IC50 values from in vitro CYP inhibition assays suggest that the methanolic extract of KF is likely to cause in vivo inhibition of all the CYPs tested and thereby potentially causing HDI (Table 3). All alkyl phenols except fatimahol are predicted to have a likely in vivo effect toward CYP2C9 and 2C19 (Table 3). As show in Table 3, the extract or the compounds did not show any strong inhibition of CYP2D6 and CYP1A2. Accordingly, we did not further determine the time-dependent inhibition (TDI) of these two enzymes.The calculated % yield and concentration per dose  was more for alkyl phenols compared to saponins from KF roots as shown in in vitro methods.There is increasing evidence that the global use of herbal supplements for the treatment of a wide spectrum of ailments has been on the rise. Concurrently, there are more clinical cases of toxicity caused by concomitant administration of herbal supplements with conventional medicines . This riPanax notoginseng were found to induce CYP1A2 with no inhibitory effect on the activity of other CYPs -pentadecenyl]resorcinol, fatimahol, and the extract was greater than positive control troleandomycin, which is considered to be a clinically relevant mechanism based inhibitor. Previous structural studies have shown that the phenolic moiety strongly binds to CYP3A4 enzyme causing a strong inhibition -pentadecenyl]resorcinol which contains the phenolic hydroxyl group . The other alkyl phenols which did not have hydroxyl group showed comparatively weaker inhibition than 5-[10(Z)-pentadecenyl]resorcinol. This TDI of CYP3A4 could be due to either mechanism based inhibition (irreversible), or due to the generation of metabolites which may cause stronger inhibition of the enzyme. Further studies in liver microsomes are needed to clarify the exact mechanism of inhibition.Time dependent inhibitors are categorized as mechanism based inhibitors. Such inhibitors are generally considered to have more profound clinical effects compared to reversible inhibitors as they form strong covalent bonds and thereby inactivate the CYPs . In ordehibition  which maf CYP3A4 . Such a 50 values below 10 μM in the digoxin transport assay are recommended for further evaluation in the in vivo system  with conventional drugs could cause a possibility of drug-herb interaction. Further studies are warranted in this direction.In conclusion, this study demonstrated that the methanolic extract of KF and its constituents strongly inhibited (TDI) a major drug metabolizing enzyme CYP3A4, while a moderate reversible inhibition was seen with CYP2C9 and 2C19 with minimal effects on CYP 2D6 and 1A2. Inhibition of P-gp and PXR by the methanol extract could be attributed to the presence of saponins while inhibition of CYPs could be due to alkyl phenols. Taken together, concomitant use of The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A receptor‐mediated inhibition: phasic or classical inhibitory postsynaptic currents (IPSCs) generated by the activation of synaptic GABAA receptors (sGABAAR) and tonic inhibition generated by extra‐ or peri‐synaptic GABAA receptors (eGABAAR) A inhibition in TC neurons has not been examined. Here, we investigated whether 5‐HT and its 5‐HT1A, 5‐HT2A and 5‐HT2C receptors exert a control over tonic and phasic GABAA currents in dLGN TC neurons. We used whole cell patch clamp recordings in coronal slices (300 mm) containing the dLGN from postnatal day 20–25 Wistar rats. Data analysis and experimental procedures were similar to those previously described A current are shown in Table A inhibition , but has no action on tonic inhibition. These effects are identical to those observed following 5‐HT1A/7R activation with 8‐OH‐DPAT. On the other hand, α‐M‐5‐HT and mCPP enhances and reduces, respectively, both phasic and tonic GABAA inhibition. These effects are dependent on 5‐HT2AR and 5‐HT2CR activation, respectively, as they are blocked by co‐perfusion with selective antagonists, ketanserin, and SB242084. Thus, the lack of 5‐HT modulation of tonic inhibition might be explained by the counterbalance of co‐activation of 5‐HT2ARs and 5‐HT2CRs by the endogenous ligand  neurons, including those of the dorsal lateral geniculate nucleus (dLGN), one of the visual sensory thalamic nuclei, exhibit two forms of GABA2ARs  1AR‐dependent suppression of protein kinase A (PKA) activity) 1A/7R activation in the dLGN.Our findings are in agreement with recent evidence in visual cortex showing that 5‐HT enhances phasic inhibition by activating 5‐HTin vivo studies in which stimulation of the dorsal raphe nucleus decreased TC neuron firing in the dLGN in vitro intracellular recordings of ferret TC cells found that 5‐HT is predominantly hyperpolarizing in all thalamic nuclei tested except for the dLGN, medial geniculate and in a subset of pulvinar neurons, in which depolarizing responses were observed 2CR activation with α‐M‐5‐HT and the highly selective ligand CP‐809,101 produced depolarization of TC neurons shifting their firing from bursts to tonic 1A/7 are present in the thalamus 2CR immunoreactivity has been detected, although not somatically, in dLGN TC neurons 2AR immunostaining did not reach detectable levels 2AR and 5‐HT2CR mRNA are expressed in the dLGN GABAergic interneurons of young rats 2Rs on dendritic F2 terminals of dLGN interneurons, as previously shown Moreover, our study is in agreement with previous 1A/7R and 5‐HT2A/2CRs might also be located postsynaptically on TC neurons, and their activation may lead to phosphorylation of different subunits of sGABAARs and eGABAARs acting to differentially modulate their function. The binding of 5‐HT to 5‐HT1A/7R and 5‐HT2A/2CRs might activate multiple signal transduction cascades, which ultimately activate different protein kinases, such as PKA or protein kinase C (PKC) and differently regulate sGABAARs and eGABAARs, as indicated by our results. On the other hand, the potential contribution of 5‐HTRs on retinal and cortical terminals can be ruled out as glutamatergic function was blocked in our preparations through the addition of kynurenic acid to the recording solution.Nevertheless, 5‐HTA current by 5‐HTRs in the thalamus and also to highlight that phasic and tonic inhibition in the dLGN are modulated by 5‐HT through different receptor subtypes, leading to a finely tuned balance of sensory information processing in the dLGN. By showing a differential modulation of phasic versus tonic GABAA inhibition, our results demonstrate a novel mechanism by which the ascending serotonergic afferents can control the thalamic gate to the visual cortex in a behavioral state‐dependent manner. Moreover, because of the putative role for thalamic tonic GABAA inhibition in sleep regulation and pathological oscillations, such as those present in absence epilepsy, the opposing effects of 5‐HT2ARs and 5‐HT2CRs activation may provide suitable targets for pharmacological intervention in sleep and other CNS disorders.Overall, this study is the first to show a modulation of tonic GABA


The P70 ribosomal protein S6 kinase 1 (P70S6K1) is activated by the mammalian target of rapamycin (mTORC1) and regulates proliferation, growth, and metabolism. PF-4708671 is a novel, cell-permeable, has been proposed to be a highly specific inhibitor of p70S6K1. It is used in micromolar concentration range to dissect signaling pathways downstream of mTORC1 and to study the function of p70S6K1. Here we show that PF-4708671 induces AMP-activated protein kinase (AMPK) phosphorylation and activation in immortalized mouse embryonic fibroblasts (MEF) independently of p70S6K1, due to specific inhibition of mitochondrial respiratory chain Complex I. P70 ribosomal protein S6 kinases (p70S6K) 1 & 2  are two isoforms of the AGC kinase (protein kinase A/protein kinase G/protein kinase C) family. The AGC kinase family consists of about 60 members that share a conserved catalytic kinase domain p70S6K isoforms are activated by the phosphorylation of Thr389 catalyzed by the rapamycin-sensitive mammalian TOR complex 1 (mTORC1) 50 of 160 nM). In a panel of ∼90 protein and lipid kinases of most closely related AGC family members, PF-4708671 was found to be highly selective for p70S6K1, being 400-fold less potent for inhibiting S6K2. Furthermore, PF-4708671 does not inhibit pyruvate dehydrogenase kinase, isozyme 1 (PDK1), which acts upstream of several AGC kinases, including p70S6K1. Among the AGC kinase family, mitogen- and stress-activated protein kinase 1 (MSK1) was found to be the next most sensitive to PF-470867, with an IC50 of 950 nM in vitro. However, PF-470867 did not appear to inhibit cellular MSK1 within the 1 to 10 micromolar concentration range, where it strongly inhibits p70S6K1 PF-4708671 is a cell-permeate p70S6K1-specific inhibitor Here we show that PF-4708671 activates AMPK in p70S6K 1 & 2 double knockout (DKO) immortalized mouse embryonic fibroblasts (MEF), implying AMPK activation independently of P70S6K1 inhibition. It does so directly by inhibiting mitochondrial respiratory chain Complex I. Since AMPK inhibits mTORC1 and p70S6K1 activities, its direct activation by PF-4708671 may amplify p70S6K inhibition by PF-4708671.Immortalized mouse embryonic fibroblasts (MEF) derived from S6K double knockout or S6K wild type cells were a kind gift of Mario Pende  MEF mitochondria were isolated by differential centrifugation: cells were homogenized in buffer A  with a Dounce homogenizer (Teflon glass) and centrifuged for 3 min at 2,000 g to remove nuclei and cell debris. The supernatant was centrifuged for 10 min at 12,000 g at 4°C, and the pellet was re-suspended in buffer A containing 0.02% digitonin (Sigma-Aldrich) and re-centrifuged. The mitochondrial pellet was washed again twice with buffer A and kept at –80°C until use.C by standard spectrophotometry, as previously described Enzymatic activities of respiratory chain complexes were measured at 37Citrate synthase (CS), a ubiquitous mitochondrial matrix enzyme serving as normalizer, was measured at 412 nm by the release of CoASH coupled to 50,50-dithiobis (2-nitrobenzoic) acid, in the presence of acetyl-CoA and oxaloacetate. Activities of mitochondrial respiratory chain complexes in the presence of added PF-4708671 are presented relative to respective activities in the presence of vehicle (DMSO). Protein concentration was determined by the Lowry method and calculated according to a bovine serum albumin (BSA) standard curve.http://www.bioind.com/page_14372) containing 5% FCS  was used as the standard medium in the XF and referred to as “assay medium”. The Seahorse platform can calculate the extracellular acidification rate (ECAR) in a non-buffered medium. Thus, because L-15 is HEPES buffered extracellular acidification rate (ECAR) was not used.L-15medium  in 500 µl complete growth medium (DMEM +10% FCS) and then incubated at 37°C/5% CO2 for 16 h, followed by discarding the growth medium and washing the cells three times with 750 µl of assay medium. The cells were further incubated at 37°C with no CO2 supplementation for 4 hours to allow temperature and pH equilibration. Prior to each measurement, the XF24 Analyzer mixed the assay media for 3 min followed by 2 minutes wait to allow for oxygen pressure equilibration. Then oxygen consumption rate (OCR) was measured for 3 min. This was repeated to establish the OCR baseline. PF-4708671 or Rapamycin were injected into each well to reach the final working concentration of 2.5/5 µM and 50 nM, respectively. DMSO prepared in assay medium was injected as control. After the compounds injection, OCR measurements were made. Responses were expressed as a percentage of the baseline rate prior to compound addition. Statistical analysis was done using Seahorse analysis software using OCR area under the curve ANOVA test, as recommended by the manufacturer.MEF cells were seeded in XF 24-well cell culture microplates (Seahorse Bioscience) at 20×10Cells were washed briefly in ice cold PBS, followed by adding lysis buffer (0.1M Tris pH6.8 containing 1% SDS). Lysate was incubated on ice for 30 minutes, and then centrifuged at 14,000 g at 4°C for 10 minutes. For Western blot analysis, 20 µg of protein extract was subjected to 8% or 10% SDS/PAGE and transferred to nitrocellulose membranes. After blocking in 5% non-fat milk, blots were incubated with AMPK (#2532), Phospho-AMPK (#2531), ACC (#3662), Phospho-ACC (#3661), P70S6K1 (#9202), Phospho-S6 ribosomal protein 240/244 (#5364), Phospho-S6 ribosomal protein 235/236 (#4858), beta-actin (#4967) antibodies for overnight, at 4°C. All antibodies were from Cell Signaling Technology. Blots were washed in Tris buffered saline - 0.2% Tween20 (TBS-T) three times, and incubated for one hour at room temperature with anti-Rabbit or anti-Mouse secondary antibody diluted 1∶350 in 5% no-fat milk . Detection was performed using chemiluminescence reagent on microchemi digital platform (DNR bio-imaging systems). Protein concentration in extracts was determined using the BCA protein reagent (Pierce).In line with previous reports Concomitantly with AMPK activation, PF-4708671 inhibited dose dependently the oxygen consumption of DKO MEF , resulti−6; Inhibition of oxygen consumption by PF-4708671 was further pursued by studying PF-4708671 effects in isolated mitochondria. Mitochondrial citrate synthase, Complex II, succinate dehydrogenase, Complex II+III, and complex IV activities were unaffected by PF-4708671 . However50 of PF-4708671 in inhibiting mitochondrial Complex I was ∼5 µM, namely, within the working concentration range of 3–10 µM used to effectively block p70S6K1 The present study describes activation of AMPK by PF-4708671, a novel cell-permeable P70S6K1 inhibitor. AMPK activation by PF-4708671 is independent of p70S6K1, and may be ascribed to mitochondrial Complex I inhibition. Thus, AMPK activation and suppression of oxygen consumption by PF-4708671 were both observed in p70S6K null cells. The calculated ICp70S6K1 is activated by phosphorylation of its Thr389 by mTORC1 In conclusion, direct activation of AMPK by PF-4708671, combined with inhibition of P70S6K1 activity, may open new prospects for PF-4708671 in modifying mTORC1 and p70S6K1 control of metabolism, protein synthesis, autophagy, and proliferation.Figure S1DKO and WT MEFs comparison. p70S6K double knockout MEF show lower levels of phospho-rpS6. Wild type MEF and p70S6K double knockout MEF were grown in complete medium. p70S6K1 and phospho-S6 (235/236 and 240/244) were determined by SDS-PAGE as described in (TIF)Click here for additional data file.Figure S2BT-474 show pACC elevation due to PF-4708671. BT-474 were treated with PF-4708671 as indicated. Phospho-ACC (Ser79), phospho- AKT (Ser473), AKT, and phospho-S6 (235/236 and 240/244) were determined by SDS-PAGE as described in (TIF)Click here for additional data file.Figure S3Densitometry of . AMPK activation by PF-4708671. Wild type MEF (A) and p70S6K double knockout MEF (B) were treated with PF-4708671 as indicated. Densitometry of Phospho-ACC (Ser79), ACC, phospho-AMPK(Thr172), AMPK, and phospho-S6 (235/236 and 240/244) were determined by chemiluminescence as described in (TIF)Click here for additional data file.Figure S4Inhibition of BT-474 oxygen consumption by PF-4708671. BT-474 were treated with vehicle (turquoise line) or 3 µM of PF-4708671(pink). Oxygen consumption rate (OCR) of was determined by the Seahorse platform as described in (TIF)Click here for additional data file.Table S1Seahorse platform and mitochondrial assays statistics. ANOVA statistical analysis of the OCR area under the curve showed that after 2.5 uM or 5 uM PF-4708671 treatment the OCR was significantly decreased (manufacturer output), and mitochondrial assay statistics.(XLSX)Click here for additional data file.


Anisakis. This article reports the gene sequencing, cloning, expression and preliminary biochemical and bioinformatically-based structural characterization of a new Anisakis serpin (ANISERP).Serine proteinase inhibitors (serpins) finely regulate serine proteinase activity via a suicide substrate-like inhibitory mechanism. In parasitic nematodes, some serpins interact with host physiological processes; however, little is known about these essential molecules in AniSerp gene was cloned by specific RACE-PCR after screening an Anisakis simplex (L3) cDNA library. For biochemical assays, the AniSerp gene was subcloned into both prokaryotic and eukaryotic vectors, and the recombinant proteins were purified. The inhibitory properties of the proteins were tested in classical biochemical assays using human serine peptidases and AMC substrates. Immunolocalization of ANISERP, theoretical structural analysis and bioinformatically-based structural modelling of the ANISERP protein were also conducted.The full AniSerp gene was found to have 1194 nucleotides, coding for a protein of 397 amino acid residues plus a putative N-terminal signal peptide. It showed significant similarity to other nematode, arthropod and mammalian serpins. The recombinant ANISERP expressed in the prokaryotic and eukaryotic systems inhibited the human serine proteases thrombin, trypsin and cathepsin G in a concentration-dependent manner. No inhibitory activity against Factor Xa, Factor XIa, Factor XIIa, elastase, plasmin or chymotrypsin was observed. ANISERP also acted on the cysteine protease cathepsin L. ANISERP was mainly localized in the nematode pseudocoelomic fluid, somatic muscle cell bodies and intestinal cells. The findings of molecular dynamics studies suggest that ANISERP inhibits thrombin via a suicide substrate-like inhibitory mechanism, similar to the mechanism of action of mammalian coagulation inhibitors. In contrast to findings concerning human antithrombin III, heparin had no effect on ANISERP anticoagulant inhibitory activity.The Anisakis regulatory serpin and that the inhibitory activity against thrombin depends on a suicide substrate-like inhibitory mechanism, similar to that described for human antithrombin (AT)-III. The fact that heparin does not modulate the anticoagulant activity of ANISERP might be explained by the absence in the latter of five of the six positively charged residues usually seen at the AT-III-heparin binding site.Our findings suggest that ANISERP is an internal  Anisakis spp. are parasitic nematodes that infect the gastrointestinal tract of sea mammals .In e family –18. The Anisakis simplex truncated cDNA that showed notable similarity to serpin genes (NCBI) was obtained by screening a cDNA library obtained from the L3 stage larvae. Amplification of the 5′ end was performed by RACE-PCR and using a parasite cDNA collection prepared with the Marathon cDNA amplification kit (Clontech) and the forward primer AP1 (5′ CTAATACGACTCACTATAGGGC 3′), which corresponds to the AP1 adaptor sequence of the A.simplex cDNA collection, and reverse primer SR1 (5′ ACCCGCAGTAGTTTTATCCATTTGTTCG 3′), the design of which was based on the truncated cDNA sequence. On the basis of the information obtained, two new primers were used to amplify the full gene by PCR: the forward primer SER5′ (5′ ATGATGACAGCATTACCGTTTTTAAC 3′) and the reverse primer SER3′ (5′ TCAGTGGAAACGACCAATAAACAGAATGCG 3′). The gene was named AniSerp. Standard amplification protocols were used in all cases -p16 [E/K/R]-p15 [G]-p14 [T/S]-p13 [X]-p12-9 [A/G/S]-p8-1 [X]-p1′-4′). Database comparisons showed similarities between ANISERP and different serine proteinase inhibitors of nematodes and other organisms. The highest similarity scores were obtained for the serpins described in Toxocaracanis , Ascarissuum . ANISERP also showed similarity to the Bostaurus serpin peptidase inhibitor , mouse (Mus musculus) serpin 3b  and mouse squamous cell carcinoma antigen 2-related protein 1 . These findings were used together to design ANISERP functional characterization experiments.The truncated E. coli system, the conditions were 0.01 mM IPTG and incubation at 16 °C for 16 h. For the baculovirus system, the conditions were a multiplicity of infection of 0.1 (virus titre 5×106pfu/ml), incubation at 27 °C and collection at 72 h.To obtain the recombinant ANISERP protein, the gene without the putative N-terminal signal peptide was subcloned into pGEX, and the full sequence was subcloned into the baculovirus vector system. These genes were expressed in prokaryotic and eukaryotic cells respectively, and they were then purified. To collect the proteins in functional form, different expression conditions were tested. For the 50 152 nM), trypsin (IC50 173 nM), cathepsin G (IC50 542 nM) and cathepsin L (IC50 217 nM) in a concentration-dependent manner , indicating that recognition was specific to ANISERP. As shown in Fig. To locate the possible site of synthesis and final destination of ANISERP, an immunohistochemical study was performed on Anisakis larvae, three sources of parasite antigens can be delivered to host tissues while larvae remain alive: i) cuticle antigens, which are in direct contact with host tissues, ii) gut antigens, which can be released through the mouth/anus, and iii) secretory antigens, which are released by the excretory cell through the excretory pore, located at the base of subventral lips; the latter constitutes the main source of known Anisakis allergens. Somatic antigens can also come into contact with host tissues, but only in cases in which larvae die and remain in tissues . The absence of staining in the dorsal oesophageal gland, intestinal lumen, excretory cell and cuticle (Table Anisakis during the acute phase (data not shown).When marine mammals or humans are infected by L3 le Table  clearly Anisakis have not been reported before, this type of activity has been reported in muscular cells of Caenorhabditis elegans transfected with a plasmid encoding a signal peptide fused to green fluorescent protein [As the strongest staining was observed in myocytons and pseudocoelomic fluid, we can hypothesize that somatic muscle cells are the major source of ANISERP, which can be then released to the haemolymph. Although the putative secretory functions of somatic muscle cells in  protein . The preAnisakis serpin is recognized by the polyclonal antibody. Although this possibility merits further investigation, the fact that other similar serpins exist in the Anisakis transcriptome (unpublished results) is consistent with the hypothesis.In addition to myocytons and pseudocoelomic fluid, staining for ANISERP was also intense in intestinal cells. As intestinal and muscular cells originate from different germinal layers (i.e. endoderm and mesoderm respectively), we can also hypothesize that more than one in silico analysis of the ANISERP molecule was performed to predict its tertiary structure and help to determine how it might interact with the serin proteases that it inhibits, based on characteristic and conserved serpin folding. Thrombin protease was chosen as a model as it yielded the lowest IC50. To obtain a reliable representation of the actual interactions between proteins, a homology-based 3D model of ANISERP bound to thrombin was generated. To obtain an even more realistic molecular model for the Anisakis serpin, the Ala205 located at the catalytic centre of human thrombin was substituted by a Ser residue (model 1JMO). On the basis of the initial interaction model, short-step (2 ns) molecular dynamics were applied to the whole structure to help the amino acid side chains and the reactive centre loop (RCL) backbone achieve a stable, low-energy conformation that would allow analysis of specific contacts between the two proteins . In contrast, HCII inhibits the serine proteases thrombin, chymotrypsin and cathepsin G. The distinct enzyme specificities of ATand HCII appear to be the result of amino acid changes in P1 and the immediately adjacent residues of the RCL-enzyme recognition sequence. AT, which has an Arg at P1, is an inhibitor of the Arg-proteinases thrombin and FXa, while HCII is a specific thrombin inhibitor with an unfavourable Leu residue at P1 . The inhAs there is some evidence , 38 thatIn an attempt to understand the lack of effect of heparin on the ANISERP anticoagulant action, the hypothetical heparin binding site residues were aligned with the AT and HCII heparin binding site residues described elsewhere . Figure Anisakis serpin that displays in vitro inhibitory activity. The inhibitory action of ANISERP against thrombin appears to depend on a suicide substrate-like inhibitory mechanism, similar to that displayed by human AT-III. The fact that heparin does not modulate the anticoagulant activity of ANISERP might be explained by the absence in its structure of five of the six positively charged residues usually seen in the AT-III-heparin binding site. The findings of the immunolocalization study indicate that ANISERP exerts its function as an internal regulatory serpin and rule out a role at the parasite-host interface.ANISERP is an internally-secreted


Activating mutations in FLT3 confer poor prognosis for individuals with acute myeloid leukemia (AML). Clinically active investigational FLT3 inhibitors can achieve complete remissions but their utility has been hampered by acquired resistance and myelosuppression attributed to a ‘synthetic lethal toxicity’ arising from simultaneous inhibition of FLT3 and KIT. We report a novel chemical strategy for selective FLT3 inhibition while avoiding KIT inhibition with the staurosporine analog, Star 27. Star 27 maintains potency against FLT3 in proliferation assays of FLT3-transformed cells compared with KIT-transformed cells, shows no toxicity towards normal human hematopoiesis at concentrations that inhibit primary FLT3-mutant AML blast growth, and is active against mutations that confer resistance to clinical inhibitors. As a more complete understanding of kinase networks emerges, it may be possible to define anti-targets such as KIT in the case of AML to allow improved kinase inhibitor design of clinical agents with enhanced efficacy and reduced toxicity.DOI:http://dx.doi.org/10.7554/eLife.03445.001 Major advances in cancer therapy have improved the treatment options for many patients. However, many cancer treatments are toxic or have severe side effects, making them difficult for patients to tolerate. One cause of these side effects is that many cancer therapies kill both normal cells and cancer cells. Developing cancer therapies that are more targeted is therefore a priority in cancer research.Acute myeloid leukemia is a type of blood cancer that has proven difficult to treat without causing serious side effects. This cancer is very aggressive and only about 1 in 4 patients are successfully cured of their cancer. At present, physicians treat acute myeloid leukemia with chemotherapy, which kills both the cancer cells and some of the patient's healthy cells.Many patients with acute myeloid leukemia have mutations in the gene encoding an enzyme called Fms-like tyrosine kinase 3 (FLT3). This mutation makes the enzyme permanently active, and patients with the mutation have a greater risk of their cancer recurring or death. Scientists have recently discovered that treatments that inhibit the FLT3 enzyme can be effective against cancer. However, the drugs investigated so far also interfere with the patient's ability to produce new blood cells, which can lead to infections or an inability to recover from bleeding. Therefore, no new drugs have yet been approved for general use.Warkentin et al. suspected the reason for the adverse effects of FLT3 inhibitors is that these drugs also inhibit another enzyme necessary for blood cell production. Previous work showed that inhibiting one or the other of the enzymes still allows blood cells to be produced as normal: it is only when both are inhibited that production problems arise. Warkentin et al. therefore looked for a chemical that inhibits only the FLT3 enzyme and found one called Star 27. Tests revealed that this inhibits FLT3 and prevents the growth and spread of cancerous cells but does not impair blood cell production. Additionally, Star 27 continues to work even when mutations arise in the cancer cells that cause resistance to other FLT3 inhibitors.The findings demonstrate that when it comes to drug development, it is sometimes as important to avoid certain molecular targets as it is to hit others. Understanding the network of enzymes that FLT3 works with could therefore help researchers to develop more effective and safer cancer treatments.DOI:http://dx.doi.org/10.7554/eLife.03445.002 Kinase inhibitors are among the fastest growing new class of therapeutics for treating cancer, with 25 new kinase inhibitors approved by the FDA in the last 14 years . Since tinternal tandem duplications (FLT3-ITD) (One disease where this anti-target  concept LT3-ITD) . The ITDFLT3-ITD was recently validated as a therapeutic target in AML . Several. Thus, normal mature hematopoietic populations can be maintained in the context of either Flt3 or Kit inhibition alone but not dual Flt3/Kit inhibition  are important regulators of normal hematopoiesis. In 1995, Lemischka and coworkers showed that mice lacking function of either Flt3 or Kit maintained overall normal populations . Howeverhibition .50 = 67 nM for p-KIT inhibition in TF-1 cells; 65% inhibition at 100 nM, in vitro) . When combined with cytotoxic agents PKC412 showed some promise, achieving a 25% CR rate, but responses were primarily incomplete recovery of peripheral blood counts  with over 90% of patients developing grade 3/4 myelosuppression . While An vitro) . This isn vitro) . These factive conformation by disrupting a hydrogen bond from D835 to S838, and thus limit the efficacy of Type II inhibitors such as AC220. We have recently proposed that a Type I inhibitor, which binds to the active kinase conformation, would circumvent these mutations that confer resistance to AC220  equals isobutyl (Star 12), AS Src kinase is potently inhibited but WT kinases remain unaffected. However, we also observed that Star 12, in a panel of 319 kinases, weakly inhibits only one WT kinase, FLT3 . Thus, the C7-alkyl group of Star 12 may allow for weak but selective inhibition of FLT3 over the anti-target KIT, which contributes to myelosuppression when FLT3 is also inhibited. Although substitution of an isobutyl group at C7 reduced potency, we hypothesized that combining the FLT3/KIT selectivity of Star 12 and the potency features of PKC412 would generate an optimal FLT3 inhibitor also capable of targeting emerging mutations  , though utations .10.7554/n-propyl amine to mimic the N-methyl amide of PKC412 produced comparable values (iso) reduced activity, further indicating a well-defined structure activity relationship , Star 23 showed improved differentiation between FLT3 and other Class III RTKs , and Star 27 proved to have the best selectivity (IC50 = <0.6–9880 nM). This selectivity increases from (a) KIT/FLT3 ratio of >72 for PKC412 to >1700 for Star 27 and (b) CSF1R/FLT3 ratio of 8 for PKC412 to >16,500 for Star 27 ; HMC1.1 cells (dependent on KIT V560G); and K562 cells (which express the BCR-ABL fusion kinase) as a control for non-KIT-related toxicity. Sorafenib, AC220, and ponatinib all displayed equipotent inhibition of FLT3 and KIT-driven cell lines  samples. One of the four donor samples is shown in Danio rerio (zebrafish) for studying hematopoiesis , finding no change in heart and tail morphology -stained in situ hybridization in the posterior blood island (PBI). Representative cross sections for the PBI for DMSO, Star 27, and PKC412 are shown . In contEfficacy in the AML FLT3-ITD context is quantified by binning the FLT3-ITD blast phenotype into Normal, Intermediate, and Severe states . Star 273 van der Waal's volume of methyl (vs. 1.17 Å3 for H). Taken together both arguments may explain the greater affinity of PKC412 for both kinases and of Star 27 for FLT3 but not KIT . This model may account for the selectivity seen for Star 27 for kinases bearing Phe GKs  and away from those bearing Thr GKs  Electronics: we calculate that PKC412 maintains a partial positive charge at its lactam C7 position, matching the partial negative charge of KIT's Thr GK , while S50 values are presented as log10-fold resistance in a color-coded heat map to highlight the range of offset. Using published IC50 values for sorafenib, AC220, and ponatinib for comparison  will dr50s for FLT3 and KIT phosphorylation . While MLN518 showed promise with reducing FLT3-ITD+ blasts in primary patient-derived samples, exposure of the drug to the BM of healthy individuals at a dose necessary for primary FLT3-ITD+ CFU reduction, led to severe depletion of healthy hematopoietic colony formation at 1 µM.A former targeted clinical candidate, tandutinib (MLN518), may further demonstrate directly the effects of the FLT3/KIT synthetic lethal toxicity model . MLN518 −/−) to be important to osteoclastogenesis  likely caused the observed decrease in toxicity.Our focus on the staurasporine scaffold maintains the beneficial aspects of PKC412's potency against drug-resistant FLT3 mutations . Since bThe strategy reported here for gaining selectivity for FLT3 inhibition over KIT and other Class III RTKs may have importance in other hematopoietic pathology as well. FLT3 has been shown to be important for classical DC maintenance and necessary for inflammatory DCs . ConsequRecent clinical experience with targeted inhibition of mutated kinases in cancer suggests that efficacy is driven by maximal pathway inhibition. In the case of vemurafenib, <80% pathway inhibition in BRAF (V600E) mutant melanoma afforded no tumor shrinkage, while >90% inhibition showed profound clinical benefit . The chaPKC412 was purchased from Selleckchem .Staralogs  were syn2, 0.2 mM DTT, 1 mg/ml BSA) to a concentration of 2 nM and pre-incubated with 2.5% (vol/vol) DMSO, 100 µM peptide (sequence EAIYAAPFKKK [Abltide]), and varying concentrations of inhibitor from 20 µM by fourths down to 1.2 nM for 10 min pre-incubation. Kinase reactions were initiated by the addition of 100 µM cold ATP supplemented with 2.5 µCi γ32P ATP per well and allowed to proceed at RT. At 15 min, 3 µL of the reactions were spotted onto phosphocellulose sheets  and subsequently soaked in wash buffer (1.0% [vol/vol] phosphoric acid) at least five times for 5 min each. The sheets were then dried, and transferred radioactivity was measured by phosphorimaging using a Typhoon scanner (Molecular Dynamics). Radioactive counts were quantified using ImageQuant software , and titration data were fit to a sigmoidal dose response to derive IC50 values using the Prism 4.0 software package. Experiments were performed 2–4 times, each in triplicate, with eight dosages, to derive standard error of the mean values. For 2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO. Compounds were delivered into the reaction, followed 20 min later by addition of a mixture of ATP  and 33P ATP (PerkinElmer) to a final concentration approximating each kinase's Km-ATP . Reactions were carried out at 25°C for 120 min, followed by spotting of the reactions onto P81 ion exchange filter paper (Whatman). Unbound phosphate was removed by extensive washing of filters in 0.75% phosphoric acid. After subtraction of background derived from control reactions containing inactive enzyme, kinase activity data were expressed as the percent remaining kinase activity in test samples compared to vehicle (dimethyl sulfoxide) reactions. IC50 values and curve fits were obtained using Prism (GraphPad Software). Data obtained in singlicate with two biological replicates and IC50 values presented ±SEM in For 0P-labeled ATP activity assay performed by Reaction Biology Corp. Assays performed at identical conditions to those of PKC412 (319 kinases were tested in a duplicate single dose (1 μM) format using a f PKC412 .Stable Ba/F3 lines were generated by retroviral spinfection with the appropriate mutated plasmid as previously described .3 cells per well) were plated in each well of a 96-well plate with 0.1 ml of RPMI 1640 + 10% (vol/vol) FCS containing the appropriate concentration of drug in triplicate, and cell viability was assessed after 48 hr as previously described  FCS for 48 hr. Cells were fixed with 4% (vol/vol) paraformaldehyde (Electron Microscopy Sciences) and permeabilized with 100% (vol/vol) methanol  followed by staining with a FITC-conjugated antiactive caspase-3 antibody . Cells were run on a BD LSRFortessa cell analyzer, and data were analyzed using FlowJo . Percentage of live cells was determined by negative staining for activated caspase-3 see .Exponentially growing Molm14, HB119, or Ba/F3 cells stably expressing mutant isoforms were plated in RPMI medium 1640 + 10% (vol/vol) FCS supplemented with PKC412 or Star 27 at the indicated concentration. HMC1.2 cells were cultured and treated in IMDM + 10% FCS. After a 90-min incubation, the cells were washed in PBS, lysed, and processed as previously described. Immunoblotting was performed using anti-phospho-FLT3, anti-phospho-KIT, anti-phospho-STAT5, anti-STAT5, anti-phospho-ERK, anti-ERK, anti-phospho-S6, anti-S6, anti-KIT  and anti-FLT3 S18 antibody .Wild-type (WT) zebrafish were maintained under standard conditions, and embryos were staged as described . The stu2) for 13–14 days. Colonies were counted individually using traditional microscopy. CFU and BFU types reported separately (CFU-GEMM colonies counted as one of each). Results shown for FLT3 ITD primary blasts  before cryopreservation in 10% (vol/vol) DMSO or FCS  as previously described . review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent , a Reviewing editor, and 3 reviewers.Thank you for sending your work entitled ”Overcoming Myelosuppression Due to Synthetic Lethal Toxicity for FLT3-Targeted Acute Myeloid Leukemia Therapy” for consideration at The Reviewing editor and the reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.The reviewers all found your manuscript to be interesting and significant. Some criticisms were also noted, however. Both Reviewers 1 and 2 expressed an interest in seeing a broader selectivity analysis for STAR27, and Reviewer 3 raised some valid points about the cellular models used to mimic in vivo hematopoiesis and whether they accurately predict in vivo toxicity, or lack thereof (see specific comments below). We would be interested in receiving a suitably revised manuscript that addresses these comments.Reviewer 1 major comment:eLife. My only suggestion is that the authors discuss what the likely kinome-wide profile of STAR27 is. The Shokat Lab has shown that bisindolyl lactams with large substituents can be very selective for Ala or Gly gatekeeper-containing kinases. In this case, they are complementing the Phe residue of FLT3 with a small methyl residue in order to obtain selectivity for FLT3 over KIT . This could potentially greatly increase the number of off-targets that this inhibitor acts upon. As STAR27 does not seem to be toxic, any off-targets do not seem to be too detrimental but it would nice if the authors speculated on how promiscuous STAR27 is based on their experience with other STAR analogs.Overall, I think the rationale for the development of a FLT3-selective inhibitor (STAR27) is sound and the authors have been successful in achieving this goal. The level of biological characterization of STAR27 is appropriate and this reagent will likely prove useful for comparing the consequences of FLT3 versus FLT3/KIT inhibition. I believe that the impact of this work is sufficient enough to warrant publication in Reviewer 2 major comment:It would have been nice to compare PKC412 and the new compound in at least a few additional kinase assays covering members outside the presented class III RTKs. Did the authors also explore the (R)-enantiomer of cpd 27?Reviewer 3 major comments:I am not sure how much stock one can take into ex vivo colony assays as a surrogate for in vivo hematopoiesis. Ideally I would like to see in vivo therapy in a mouse or xeno model, or at worst ex vivo treatment of human cells f/b transplant into NSG mice comparing the different drugs. These sorts of studies would increase the strength of these findings.What is the data that Kit inhibition is responsible for the hematopoietic toxicity? Dasatinib inhibits Kit at clinically relevant concentrations, as does imatinib, without severe cytopenias. Is the toxicity due to dual flt3/kit inhibition? If so can the authors show that adding a selective Kit inhibitor to their Flt3 inhibitor results in inhibition of colony formation? Reviewer 1 major comment:Overall, I think the rationale for the development of a FLT3-selective inhibitor (STAR27) is sound and the authors have been successful in achieving this goal. The level of biological characterization of STAR27 is appropriate and this reagent will likely prove useful for comparing the consequences of FLT3 versus FLT3/KIT inhibition. I believe that the impact of this work is sufficient enough to warrant publication in eLife. My only suggestion is that the authors discuss what the likely kinome-wide profile of STAR27 is. The Shokat Lab has shown that bisindolyl lactams with large substituents can be very selective for Ala or Gly gatekeeper-containing kinases. In this case, they are complementing the Phe residue of FLT3 with a small methyl residue in order to obtain selectivity for FLT3 over KIT . This could potentially greatly increase the number of off-targets that this inhibitor acts upon. As STAR27 does not seem to be toxic, any off-targets do not seem to be too detrimental but it would nice if the authors speculated on how promiscuous STAR27 is based on their experience with other STAR analogs.We performed a kinome-wide profiling of Star 27 new . The broReviewer 2 major comment:It would have been nice to compare PKC412 and the new compound in at least a few additional kinase assays covering members outside the presented class III RTKs. Did the authors also explore the (R)-enantiomer of cpd 27?We did not test this in this study because our previous work showed that this enantiomer lost all activity see . Our modReviewer 3 major comments:I am not sure how much stock one can take into ex vivo colony assays as a surrogate for in vivo hematopoiesis. Ideally I would like to see in vivo therapy in a mouse or xeno model, or at worst ex vivo treatment of human cells f/b transplant into NSG mice comparing the different drugs. These sorts of studies would increase the strength of these findings.To assess toxicity and efficacy in vivo we initiated a collaboration with Prof. Leung, from the University of Hong Kong, who has developed a recently validated zebrafish model for studying FLT3-ITD-driven AML, and recapitulates findings in the clinic . ZebrafiWhat is the data that Kit inhibition is responsible for the hematopoietic toxicity?KIT inhibition alone is not toxic, but in the context of KIT + FLT3 inhibition, both genetic evidence (double knockout—Lemischka, Dasatinib inhibits Kit at clinically relevant concentrations, as does imatinib, without severe cytopenias. Is the toxicity due to dual flt3/kit inhibition? If so can the authors show that adding a selective Kit inhibitor to their Flt3 inhibitor results in inhibition of colony formation?We were able to harvest normal bone marrow cells from a donor and tried combining multiple doses of Star 27 with multiple doses of either imatinib or dasatinib in the same colony assay format as we used in original