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Reaction of SnMe2Cl2 with [Li(Ph2pz)] (Ph2pz = 3,5-diphenylpyrazolate) in diethyl ether in a 1:2 ratio afforded the new transmetallation reagent [SnMe2(Ph2pz)2]. Treatment of [SnMe2(Ph2pz)2] with Ln metals in either 1,2-dimethoxyethane (dme) or tetrahydrofuran (thf) provided a new route to the divalent [Ln(Ph2pz)2(dme)2] (Ln = Sm, Eu, Yb) and the trivalent [La(Ph2pz)3(dme)2] and [Yb(Ph2pz)3(thf)2] complexes in comparable yields to those from alternative methods. These reactions enabled isolation of the highly reactive [Sm(Ph2pz)2(dme)2] complex in good yield, in addition to the new cis isomer of [Eu(Ph2pz)2(dme)2], thereby establishing a rare case of geometric isomerism in lanthanoid chemistry. 1H and 119Sn NMR spectral studies and EDAX measurements were performed on the reaction mixture and/or residue to identify the tin-containing products and suggest formation of SnMe4, Sn2Me6, tin metal and two other SnMe3 species, which plausibly result from decomposition of unstable "SnMe2". Treatment of [Yb(Ph2pz)2(dme)2] with [SnMe2(Ph2pz)2] or [Tl(Ph2pz)] resulted in the isolation of [Yb(Ph2pz)3(thf)2]·2C6D6 and [Yb(Ph2pz)3(dme)]·0.5dme complexes, the structures of which have eight-coordinate Yb atoms and contrast nine-coordination observed for previous [Ln(Ph2pz)3(thf or dme)n] complexes.

Kinesin-1 is a molecular transporter that trafficks along microtubules. There is some evidence that kinesin-1 targets specific cellular sites, but it is unclear how this spatial regulation is achieved. To investigate this process, we used a combination of in vivo imaging of kinesin heavy-chain Kif5c (an isoform of kinesin-1) fused to GFP, in vitro analyses and mathematical modelling. GFP-Kif5c fluorescent puncta localised to a subset of microtubules in live cells. These puncta moved at speeds of up to 1 μm second–1 and exchanged into cortically labelled clusters at microtubule ends. This behaviour depended on the presence of a functional motor domain, because a rigor-mutant GFP-Kif5c bound to microtubules but did not move along them. Further analysis indicated that the microtubule subset decorated by GFP-Kif5c was highly stable and primarily composed of detyrosinated tubulin. In vitro motility assays showed that the motor domain of Kif5c moved detyrosinated microtubules at significantly lower velocities than tyrosinated (unmodified) microtubules. Mathematical modelling predicted that a small increase in detyrosination would bias kinesin-1 occupancy towards detyrosinated microtubules. These data suggest that kinesin-1 preferentially binds to and trafficks on detyrosinated microtubules in vivo, providing a potential basis for the spatial targeting of kinesin-1-based cargo transport.

Two ytterbium(II) bis(phenolate) complexes, [LRYb] where R = NMe21 and OMe 2, have been synthesized and characterized, with 1 being structurally defined to be a dimeric species with an unsymmetrical coordination of the bis(phenolate) ligand which is preserved in solution. Both 1 and 2 have been oxidized by a variety of oxidants (AgX, ROH) to form heteroleptic ytterbium(III) bis(phenolate) complexes: [(LNMe2)YbPF6] (3), [(LNMe2)YbOSO2CF3(thf)] (4), [(LNMe2)YbOBut] (5), [(LNMe2)YbOPh] (6), [(LOMe)YbOPh] (7). Compound 4 has been structurally characterized as having a quasi-octahedral environment around ytterbium, with significant inter species hydrogen bonding between CHx and triflate fluorine atoms. Ligand exchange between Yb(N(SiMe3)2)3(thf)2 and H2LR yielded [(LNMe2)YbN(SiMe3)2] (8) and [(LOMe)YbN(SiMe3)2] (9), while metathesis from YbI2(thf)2 and K2LOMe reproducibly afforded the surprising oxidized product [(LOMe)2YbK(dme)2] (10), which was structurally characterized as having a distorted octahedral environment around the ytterbium(III) centre. Compounds 1–9 were used to polymerize e-caprolactone at room temperature in toluene, with only compounds 1, 2, 8 and 9 exhibiting significant catalytic activities. The polycaprolactone formed in these reactions was generally of high molecular weight and polydispersities

Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole-genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis and ciliopathy genes, including 44 components of the ubiquitin-proteasome system, 12 G-protein-coupled receptors, and 3 pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localize to the connecting cilium, and PRPF8- and PRPF31-mutated cells have ciliary defects. Combining the screen with exome sequencing data identified recessive mutations in PIBF1, also known as CEP90, and C21orf2, also known as LRRC76, as causes of the ciliopathies Joubert and Jeune syndromes. Biochemical approaches place C21orf2 within key ciliopathy-associated protein modules, offering an explanation for the skeletal and retinal involvement observed in individuals with C21orf2 variants. Our global, unbiased approaches provide insights into ciliogenesis complexity and identify roles for unanticipated pathways in human genetic disease.

Objective Ciliopathies are a group of heterogeneous disorders caused by mutations in proteins associated with primary cilia. Many proteins that are mutated in ciliopathies Joubert syndrome (JBTS), Meckel-Gruber syndrome (MKS) and nephronophthisis (NPHP) are localized to the transition zone (TZ), a compartment of the proximal region of the cilium. In particular, a protein complex known as the “MKS-JBTS module” contains many transmembrane proteins (TMEMs) that are mutated in these conditions. Here, we aim to understand the role of the ciliary proteins TMEM67, TMEM138, TMEM216, TMEM237, TMEM17 and TMEM231 by characterizing their biochemical functions and potential interactions. We hypothesize that pathogenic missense mutations disrupt the putative TMEM complex or localization to the TZ. Furthermore, we aim to identify new interacting proteins of TMEMs, including potential ligands of the orphan receptor TMEM67.

The adenomatous polyposis coli (APC) tumor suppressor protein is mutated in most colorectal carcinomas. In addition to its role in WNT signaling it is proposed to be involved in both cell migration and mitosis. Although a variety of studies have shown an APC localization along lateral membranes of adjacent epithelial cells the existence of a cortical APC localization in mammalian cells remains controversial. To address this we have used matched rat epithelial (NRK-52E) and fibroblast (NRK-49F) cell lines to investigate the localization of APC. Subconfluent cultures of NRK-52E and -49F cells displayed microtubule-associated APC populations by immunostaining. However, confluent NRK-52E, but not -49F monolayers, exhibited a cortical APC distribution. Cortical APC localized in close proximity to a number of cell junction proteins in a microtubule-independent manner while calcium switch experiments suggested that APC was recruited to the cortex only when junction assembly was complete. Confluent NRK-49F and -52E cells also showed contrasting APC localizations in response to monolayer wounding. Our data suggests APC cortical localization is a feature of confluent epithelioid cells and that the subcellular distribution of APC is therefore dependent upon both cell type and context. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc.

Use of a non-contact infrared laser (IRL) or acid Tyrode's for zona drilling before embryo biopsy was compared by assessing blastomere viability using various fluorescent markers or culture of the single biopsied blastomere, and, by cytoskeletal and molecular cytogenetic analysis of the biopsied embryos following culture to the blastocyst stage. There was no significant difference in the proportion of biopsied embryos that showed no damage in both the biopsied blastomere and in the remaining embryo (acid Tyrode's: 75% versus IRL: 68%), or in the proportion of single biopsied blastomeres that divided in culture (P > 0.05). However, single biopsied blastomeres from laser drilled embryos showed a greater tendency to form miniblastocysts. The proportion of laser or acid Tyrode's biopsied embryos that reached the blastocyst stage by day 6 was similar, although evident earlier (day 5) in the laser biopsied embryos. Spindle abnormalities at the blastocyst stage included tripolar and tetrapolar spindles, but their incidence was not significantly different from controls. In addition, no significant difference was observed in the incidence of chromosomal abnormalities and mosaicism between the two groups. It is concluded that using an IRL at a safe working distance does not cause adverse immediate or longer term effects on the development of human biopsied embryos, although damage can occur if drilling within this distance is unavoidable. Acid Tyrode's drilling can also cause damage, and tended to retard blastocyst development.

Sodium and potassium tetrakis(3,5-di-tert-butylpyrazolato)lanthanoidate(III) complexes [M[Ln(tBu(2)pz)(4)]] have been prepared by reaction of anhydrous lanthanoid trihalides with alkali metal 3,5-di-tert-butylpyrazolates at 200-300 degrees C, and a 1,2,4,5-tetramethylbenzene flux for M=K. On extraction with toluene (or occasionally directly from the reaction tube) the following complexes were isolated: [Na(PhMe)[Ln(tBu(2)pz)(4)]] (1 Ln; 1 Ln=1 Tb, 1 Ho, 1 Er, 1 Yb), [K(PhMe)[Ln(tBu(2)pz)(4)]].2 PhMe (2 Ln; 2 Ln=2 La, 2 Sm, 2 Tb, 2 Ho, 2 Yb, 2 Lu), [Na[Ln(tBu(2)pz)(4)]](n) (3 Ln; 3 Ln=3 La, 3 Tb, 3 Ho, 3 Er, 3 Yb), [K[Ln(tBu(2)pz)(4)]](n) (4 Ln; 4 Ln=4 La, 4 Nd, 4 Sm, 4 Tb, 4 Ho, 4 Er, 4 Yb, 4 Lu), with the last two classes generally being obtained by loss of toluene from 1 Ln or 2 Ln, and [Na(tBu(2)pzH)[Ln(tBu(2)pz)(4)]].PhMe (5 Ln; 5 Ln=5 Nd, 5 Er, 5 Yb). Extraction with 1,2-dimethoxyethane (DME) after isolation of 2 Ho yielded [K(dme)[Ho(tBu(2)pz)(4)]] (6 Ho). X-ray crystal structures of 1 Ln (=1 Tb, 1 Ho; P2(1)/c), 2 Ln (=2 La, 2 Sm, 2 Tb, 2 Yb, 2 Lu; Pnma), 3,4 Ln (=3 La, 3 Er, 4 Sm; P2(1)/m), and 5 Ln (=5 Nd, 5 Er, and 5 Yb; P1) show each group to be isomorphous regardless of the size of the Ln(3+) ion. All complexes contain eight-coordinate [Ln(eta(2)-tBu(2)pz)(4)] units. These are further linked to the alkali metal by bridging through two (1,2,5 Ln) or three (3,4 Ln) tBu(2)pz groups which show striking coordination versatility. Sodium is coordinated by an eta(4)-PhMe, a micro-eta(2):eta(2)-tBu(2)pz, and a micro-eta(4)(Na):eta(2)(Ln)-tBu(2)pz ligand in 1 Ln, and by one eta(1)-tBu(2)pzH and two micro-eta(3)(Na):eta(2)(Ln) ligands in 5 Ln. By contrast, potassium has one eta(6)-PhMe and two micro-eta(5)(K):eta(2)(Ln) ligands in 2 Ln. Classes 3,4 Ln form polymeric chains with the alkali metal bonded by two micro-eta(3)(NNC-M):eta(2)(Ln)-tBu(2)pz ligands within [MLn(tBu(2)pz)(4)] units which are joined together by eta(1)(C)-tBu(2)pz-Na, K linkages.

During mitosis, the spindle checkpoint delays the onset of anaphase until all chromosomes have attached properly to the mitotic spindle, preventing chromosome missegregation. BUB (budding uninhibited by benzimidazole) 1 is one of the key components of this checkpoint. BUB1 mutations are rare in cancer tissues and no mutations have been identified in gastric cancer. In mice, immunodepletion of BUB1 abolished the spindle checkpoint. Thus, aberrant expression of BUB1 protein could impair mitotic checkpoint function, resulting in aneuploidy, a common phenomenon in gastric cancer. In the present study, an antibody was generated against BUB1 and its expression was studied in gastric cancer tissue sections (n = 80) by immunohistochemistry. Nuclear BUB1 expression was found in all gastric cancer cases. The proportion of tumour cells expressing BUB1 was significantly greater in diffuse-type than in intestinal-type gastric carcinoma (p < 0.001). No correlation was found between BUB1 expression and deoxyribonucleic acid (DNA) ploidy, microsatellite instability or any other histopathological parameters investigated. To the authors' knowledge, this is the first study of BUB1 protein expression in gastric cancer tissues. Different BUB1 protein expression levels in intestinal- and diffuse-type gastric cancer may provide further evidence of a potential link between different genetic pathways and morphological phenotype in gastric carcinogenesis. However, further studies are needed to establish whether there is an association between BUB1 protein expression level and mitotic spindle checkpoint function in gastric cancer.

Studies of the ring-opening polymerization (ROP) of L-lactide by Ph2SnX2 reveal that X (X=NMe2 or OPri) influences not only the rate of polymerization but also the nature of the polymer formed. Gel permeation chromatography (GPC), mass spectral analysis and 1H NMR spectroscopy of the polymers from these reactions revealed that for X=NMe2, a bimodal molecular weight distribution of polylactide (PLA) is obtained. This is indicative of both cycles and long chains (24 kDa) being formed by both intra- and interchain transesterification. This extensive transesterification results in regeneration of the Ph2Sn[OCHMeC(O)NMe2]2 species as the resting state of the tin(IV) species. Conversely, for X=OPri only a unimodal molecular weight distribution is observed in addition to Ph2Sn[OCHMeC(O)OPri]2, which is indicative of only interchain transesterification. Similar observations were seen in studies of ROP of L-lactide by Ph3SnX precursors.

When myoblasts fuse into myotubes, the organisation of the cytoskeleton changes dramatically. For example, microtubules emanate in a radial array form the centrosome in myoblasts, but form linear arrays not linked to a centrosome in myotubes. It is not clear how these linear arrays are formed and nucleated. They could arise in a number of ways: by nucleation and release from centrosomal like structures, cytoplasmic assembly, breakage/severing or nucleation from non-centrosomal sites. To test which of the above mechanisms or combination of mechanisms are responsible we investigated the re-formation of microtubules after depolymerisation by nocodazole, using antibodies against pericentrin, γ-tubulin, EB1, and tyrosinated α-tubulin. In myoblasts, we found that when microtubules were allowed to recover after complete depolymerisation with nocodazole, microtubule recovery began within 1 min and was complete after 5 min. Microtubules grew out from the centrosome, which was positively stained for γ-tubulin or pericentrin. In untreated myotubes, microtubules were arranged in linear arrays, with EB1 at their ends. The pericentriolar protein, pericentrin was arranged in a band around the nucleus as well as discrete spots in the cytoplasm. In contrast, the microtubule nucleating protein γ-tubulin was not found in a band around the nucleus, but was found in several punctuate spots throughout the cytoplasm. Further, when microtubules were allowed to recover, after complete depolymerisation with nocodazole, recovery was not as rapid as that seen in myoblasts, and we found that regrowth began with the formation of short microtubule fragments throughout the cytoplasm. γ-tubulin was associated with these fragments. These results suggest that in myotubes, nucleation of microtubules can be non-centrosomal.

Ph3SnX reacts with L-lactide in the order X=NMe2∼OMe≫OPri>OBut in the initial ring-opening event. The rate of ring-opening of methyl substituted 1,4-dioxane-2,5-diones decreases with methyl substitution and ring-opening of 3,3,6,6-tetramethyl-1,4-dioxane-2,5-dione is not observed. From studies of the reaction between Ph3SnOPri and L-lactide the activation parameters ΔH≠=13(1) kcal mol−1 and ΔS≠=−37(3) eu have been determined. Ph3SnNMe2 reacts with cyclic esters and propylene carbonate at low temperatures to give isolable ring-opened products. The compound Ph3Sn[OCHMeC(O)OCHMeC(O)X] (where X=NMe2, OMe) are labile in solution at room temperature, yielding Ph3Sn[OCHMeC(O)X] and Ph3Sn[OCHMeC(O)]nX, where n≥3, by transesterification, along with other minor products due to phenyl/OR group transfer (i.e., Ph4Sn). Ph2Sn(NMe2)2 and L-lactide react to give Ph2Sn[OCHMeC(O)NMe2]2 by ring-opening of L-lactide followed by rapid amidation. A related compound, Ph2Sn[OCHMeC(O)OPri]2, is also formed in the reaction between Ph2Sn(OPri)2 and L-lactide but is more labile toward further ring-opening of L-lactide.

Cytosolic phospholipase A(2)-alpha (cPLA(2)-alpha) is a calcium-sensitive enzyme involved in receptor-mediated eicosanoid production. In resting cells, cPLA(2)-alpha is present in the cytosol and nucleus and translocates to membranes via its calcium-dependent lipid-binding (CaLB) domain following stimulation. cPLA(2)-alpha is also regulated by phosphorylation on several residues, which results in enhanced arachidonic acid release. Little is known about the factors controlling the nuclear localisation of cPLA(2)-alpha. Here the nuclear localisation of cPLA(2)-alpha in the EA.hy.926 human endothelial cell line was investigated. Nuclear localisation was dependent on proliferation, with subconfluent cells containing higher levels of nuclear cPLA(2)-alpha than contact-inhibited confluent or serum-starved cells. The broad-range protein kinase inhibitor staurosporine caused a decrease in the nuclear level of cPLA(2)-alpha, whereas the protein phosphatase inhibitor okadaic acid increased the level of nuclear cPLA(2)-alpha. Using inhibitors for specific mitogen-activated protein (MAP) kinases, both p42/44(MAPK) and p38(MAPK) were shown to be important in modulating nuclear localisation. Finally, inhibition of nuclear import and export using Agaricus bisporus lectin and leptomycin B, respectively, demonstrated that cPLA(2)-alpha contains functional nuclear localisation and export signals. Thus we have identified a novel mode of regulation of cPLA(2)-alpha. This, together with the increasing body of evidence supporting the role of nuclear lipid second messengers in gene expression and proliferation, may have important implications for controlling the growth of endothelial cells in angiogenesis and tumour progression.

EB1 is a microtubule tip–associated protein that interacts with the APC tumor suppressor protein and components of the dynein/dynactin complex. We have found that the C-terminal 50 and 84 amino acids (aa) of EB1 were sufficient to mediate the interactions with APC and dynactin, respectively. EB1 formed mutually exclusive complexes with APC and dynactin, and a direct interaction between EB1 and p150Gluedwas identified. EB1-GFP deletion mutants demonstrated a role for the N-terminus in mediating the EB1-microtubule interaction, whereas C-terminal regions contributed to both its microtubule tip localization and a centrosomal localization. Cells expressing the last 84 aa of EB1 fused to GFP (EB1-C84-GFP) displayed profound defects in microtubule organization and centrosomal anchoring. EB1-C84-GFP expression severely inhibited microtubule regrowth, focusing, and anchoring in transfected cells during recovery from nocodazole treatment. The recruitment of γ-tubulin and p150Gluedto centrosomes was also inhibited. None of these effects were seen in cells expressing the last 50 aa of EB1 fused to GFP. Furthermore, EB1-C84-GFP expression did not induce Golgi apparatus fragmentation. We propose that a functional interaction between EB1 and p150Gluedis required for microtubule minus end anchoring at centrosomes during the assembly and maintenance of a radial microtubule array.

Purpose The aim of this study was to investigate the interaction and co-localization of novel interacting proteins with the Leber congenital amaurosis (LCA) associated protein aryl hydrocarbon receptor interacting protein-like 1 (AIPL1). Methods The CytoTrapXR yeast two-hybrid system was used to screen a bovine retinal cDNA library. A novel interaction between AIPL1 and members of the family of EB proteins was confirmed by directed yeast two-hybrid analysis and co-immunoprecipitation assays. The localization of AIPL1 and the EB proteins in cultured cells and in retinal cryosections was examined by immunofluorescence microscopy and cryo-immunogold electron microscopy. Results Yeast two-hybrid (Y2H) analysis identified the interaction between AIPL1 and the EB proteins, EB1 and EB3. EB1 and EB3 were specifically co-immunoprecipitated with AIPL1 from SK-N-SH neuroblastoma cells. In directed 1:1 Y2H analysis, the interaction of EB1 with AIPL1 harbouring the LCA-causing mutations A197P, C239R and W278X was severely compromised. Immunofluorescent confocal microscopy revealed that AIPL1 did not co-localize with endogenous EB1 at the tips of microtubules, endogenous EB1 at the microtubule organising centre following disruption of the microtubule network, or with endogenous β-tubulin. Moreover, AIPL1 did not localize to primary cilia in ARPE-19 cells, whereas EB1 co-localized with the centrosomal marker pericentrin at the base of primary cilia. However, both AIPL1 and the EB proteins, EB1 and EB3, co-localized with centrin-3 in the connecting cilium of photoreceptor cells. Cryo-immunogold electron microscopy confirmed the co-localization of AIPL1 and EB1 in the connecting cilia in human retinal photoreceptors. Conclusions AIPL1 and the EB proteins, EB1 and EB3, localize at the connecting cilia of retinal photoreceptor cells, but do not co-localize in the cellular microtubule network or in primary cilia in non-retinal cells. These findings suggest that AIPL1 function in these cells is not related to the role of EB proteins in microtubule dynamics or primary ciliogenesis, but that their association may be related to a specific role in the specialized cilia apparatus of retinal photoreceptors.

The interaction between the adenomatous polyposis coli (APC) tumour suppressor and the microtubule-associated protein EB1 was examined. Immunoprecipitation suggested that APC and EB1 were not associated in cultures of HCT116 cells arrested in mitosis. The C-terminal 170 amino acids of APC, purified as a bacterial fusion protein, precipitated EB1 from cell extracts, significantly refining the location of the EB1 interaction domain in APC. In vitro phosphorylation of this fusion protein by either protein kinase A or p34cdc2 reduced its ability to bind to EB1. Expression of GFP fusions to C-terminal APC sequences lacking or including the APC basic domain but encompassing the EB1 binding region in SW480 cells revealed a microtubule tip association which co-localized with that of EB1. Expression of the basic domain alone revealed a non-specific microtubule localization. In vitro interaction studies confirmed that the APC basic domain did not contribute to EB1 binding. These findings strongly suggest that the interaction between APC and EB1 targets APC to microtubule tips, and that the interaction between the two proteins is down-regulated during mitosis by the previously described mitotic phosphorylation of APC.

[Tl(Ph2pz)] (Ph2pz = 3,5-diphenylpyrazolate) crystallises in two forms. The first from benzene has trinuclear molecules [Tl3(Ph2pz)3] 1 linked by intermolecular Tl–π-η6-Ph contacts. Within 1 the three thallium atoms are different, having coordination numbers of two (but also with the Tl⋯η6-Ph contact), three and four, whilst μ-η1∶η1, μ3-η1∶η2∶η1 and (the new) μ3-η1∶η1∶η1 modes of Ph2pz coordination are observed. Crystallisation of [Tl(Ph2pz)] from 1,2-dimethoxyethane (dme) gives tetranuclear molecules [Tl4(Ph2pz)4] 2 solvated by dme. Three or four coordinate Tl atoms are observed with μ3-η1∶η2∶η1 and μ3-η1∶η1∶η1-pyrazolate coordination. In addition there is an intermolecular Tl–π-η3-Ph interaction linking the tetranuclear molecules into a polymer, and sundry intramolecular Tl⋯C contacts, the most important of which is an unsymmetrical Tl–π-η5-Ph2pz interaction. Crystallisation of [Tl(Ph2pz)] or [Tl(MePhpz)] (MePhpz = 3-methyl-5-phenylpyrazolate) from dichloromethane afforded the partially hydrolysed tetranuclear cages [Tl4(Ph2pz)3(OH)] 3 or [Tl4(MePhpz)3(OH)] 4, which associate to give a dimer and a polymer respectively owing to Tl⋯Tl interactions, supported for the dimer by an intercage distant Tl–π-η5-Ph2pz contact. Complex 3 features three and four coordinate Tl atoms and μ3-η1∶η2∶η1 and μ3-η1∶η1∶η1-pyrazolate binding, whilst 4 has three, four and five coordinate thallium atoms and solely μ3-η1∶η2∶η1-pyrazolate ligands.

The complexes [Yb(bind)2(DME)2], [Yb(MePhpz)2(DME)2], [Yb(azin)2(DME)2], and [Eu(Ph2pz)2(DME)2] (bindH = 4,5-dihydro-2H-benz[g]indazole; MePhpzH = 3-methyl-5-phenylpyrazole; azinH = 7-azaindole; Ph2pzH = 3,5-diphenylpyrazole; DME = 1,2-dimethoxyethane) have been prepared by redox transmetallation between ytterbium or europium metal and the corresponding thallium(I) pyrazolate in tetrahydrofuran (THF) in the presence of mercury metal, followed by work up with DME. The thallium reagents were obtained by treatment of the appropriate pyrazole with thallium(I) ethoxide. Both [Yb(Ph2pz)2(DME)2] and [Sm(Ph2pz)2(DME)2] have been prepared by metathesis from LnI2(THF)2 and K(Ph2pz) in THF, whilst the former has also been obtained by redox transmetallation from [Hg(Ph2pz)2] and ytterbium metal and by reaction of 3,5-diphenylpyrazole with Yb(C6F5)2, and the latter from protolysis of [Sm{N(SiMe3)2}2(THF)2] with Ph2pzH, followed in each case by crystallisation of the crude product from DME. Europium(II) 3,5-di-tert-butylpyrazolate was synthesised by a redox transmetallation/ligand exchange reaction between europium metal chunks, diphenylmercury(II), and 3,5-di-tert-butylpyrazole (tBu2pzH) in the presence of mercury metal in THF, and [Eu(tBu2pz)2(DME)2] was isolated on crystallisation of the crude product from DME. The X-ray crystal structures of [Ln(L)2(DME)2] (Ln =Yb, L = bind or azin; Ln = Eu or Sm, L = Ph2pz; Ln = Eu, L = tBu2pz), each of a different crystallographic form, reveal eight-coordinate lanthanoid complexes with two η2-pyrazolate and two chelating DME ligands, but the structures differ in the relationship (cisoid or transoid) between the pyrazolate ligands. Thus cen–Ln–cen (cen = centre of the N–N bond) angles of 106.7° [Yb(bind)2(DME)2] (3a), 141.4° [Yb(azin)2(DME)2] (3c), 142.2° [Eu(Ph2pz)2(DME)2] (4a), 107.4° [Eu(tBu2pz)2(DME)2] (4b), and 102.1° [Sm(Ph2pz)2(DME)2] (5) are observed.

The fate of herpes simplex virus 1 (HSV-1) tegument proteins during infection in Vero cells was investigated immunochemically. Input virion-associated VP13/14 and VP16 localized to the nucleus early in infection, while VP1/2 localized to the nuclear envelope of the cell and VP22 could not be detected using monoclonal antibody P43. Western blotting suggested that virion-associated VP13/14, VP16 and VP22 were stable in infected cells whereas VP1/2 appeared to be processed or modified. Further studies showed that P43 recognized a phosphorylation-sensitive epitope in VP22 and suggested that virion-associated VP22 was phosphorylated upon entry to the cell. VP13/14 and VP16 were easily extracted from cells early in infection whereas VP22 was largely insoluble. Phosphatase treatment of soluble extracts caused a shift in the molecular mass of VP16 showing it was phosphorylated. As infection progressed VP16 was observed in discrete nuclear compartments where it co-localized with ICP8 and the capsid-associated protein VP22a. VP13/14 was also observed in the nucleus. P43 immunostaining appeared around 6 h post-infection as punctate nuclear foci which often localized to the edge of VP16-immunoreactive areas. Punctate P43 cytoplasmic staining appeared around 12 h post-infection. By 18 h the nuclear pattern had disappeared and an extensive cytoplasmic stain was observed which closely overlapped that of other tegument proteins. On the basis of these data we suggest that virion-associated VP22 is phosphorylated upon entry of the virus into the cell and that unphosphorylated VP22, which is preferentially recognized by P43, becomes available later in infection, initially in the nucleus, for packaging into virions.

Autosomal recessive primary microcephaly is a potential model in which to research genes involved in human brain growth. We show that two forms of the disorder result from homozygous mutations in the genes CDK5RAP2 and CENPJ. We found neuroepithelial expression of the genes during prenatal neurogenesis and protein localization to the spindle poles of mitotic cells, suggesting that a centrosomal mechanism controls neuron number in the developing mammalian brain.

Premature chromosome condensation (PCC) is a consequence of early mitotic entry, where mitosis begins before completion of DNA replication. Previously we have identified mutations in MCPH1, a DNA damage response and potential tumor suppressor gene, as a cause of primary microcephaly and PCC. Here we describe a high-throughput assay to identify modifiers of PCC. Reverse transfection of control siRNA followed by a forward transfection of MCPH1 small interfering RNA (siRNA) was performed to induce PCC. Condensin II subunits CAPG2 and CAPH2 were validated as PCC modifiers and therefore positive controls. Cell nuclei were detected by DAPI staining using an Operetta imaging system. PCC and nuclei number were determined using Columbus analysis software. Two batches of nine plates were used to determine assay efficacy. Each plate contained four negative (nontargeting) and eight positive control siRNAs. Mean % PCC was 12.35% (n = 72) for negative controls and 4.25% (n = 144) for positive controls. Overall false-positive and false-negative rates were 0% (n = 72) and 2.1% (n = 144), respectively. This assay is currently being used to screen a human druggable genome siRNA library to identify novel therapeutic targets for cancer treatment. The assay can also be used to identify novel compounds and genes that induce PCC.

Bruning-Richardson, Anke/0000-0002-9862-9805; Mertens, Peter/0000-0002-3438-3738 WOS:000327877200004 PubMed: 24165208 Background: Bluetongue virus (BTV) is an arbovirus that is responsible for 'bluetongue', an economically important disease of livestock. Although BTV is well characterised at the protein level, less is known regarding its interaction with host cells. During studies of virus inclusion body formation we observed what appeared to be a large proportion of cells in mitosis. Although the modulation of the cell cycle is well established for many viruses, this was a novel observation for BTV. We therefore undertook a study to reveal in more depth the impact of BTV upon cell division. Methods: We used a confocal microscopy approach to investigate the localisation of BTV proteins in a cellular context with their respective position relative to cellular proteins. In addition, to quantitatively assess the frequency of aberrant mitosis induction by the viral non-structural protein (NS) 2 we utilised live cell imaging to monitor HeLa-mCherry tubulin cells transfected with a plasmid expressing NS2. Results: Our data showed that these 'aberrant mitoses' can be induced in multiple cell types and by different strains of BTV. Further study confirmed multiplication of the centrosomes, each resulting in a separate mitotic spindle during mitosis. Interestingly, the BTV NS1 protein was strongly localised to the centrosomal regions. In a separate, yet related observation, the BTV NS2 protein was co-localised with the condensed chromosomes to a region suggestive of the kinetochore. Live cell imaging revealed that expression of an EGFP-NS2 fusion protein in HeLa-mCherry tubulin cells also results in mitotic defects. Conclusions: We hypothesise that NS2 is a microtubule cargo protein that may inadvertently disrupt the interaction of microtubule tips with the kinetochores during mitosis. Furthermore, the BTV NS1 protein was distinctly localised to a region encompassing the centrosome and may therefore be, at least in part, responsible for the disruption of the centrosome as observed in BTV infected mammalian cells. BBSRC and DefraUK Research & Innovation (UKRI)Biotechnology and Biological Sciences Research Council (BBSRC); Biotechnology and Biological Sciences Research CouncilUK Research & Innovation (UKRI)Biotechnology and Biological Sciences Research Council (BBSRC) [BBS/E/I/00001720] Funding Source: researchfish The authors are grateful for the funding provided by the BBSRC and Defra.

Highly aneuploid tumours are common in epithelial ovarian cancers (EOC). We investigated whether NuMA expression was associated with this phenomenon. NuMA protein levels in normal and tumour tissues, ovarian cell lines and primary cultures of malignant cells derived from ovarian ascitic fluids were analysed by Affymetrix microarray analysis, immunoblotting, immunohistochemistry (IHC) and immunofluorescence (IF), with results correlated to associated clinical data. Aneuploidy status in primary cultures was determined by FACS analysis. Affymetrix microarray data indicated that NuMA was overexpressed in tumour tissue, primary cultures and cell lines compared to normal ovarian tissue. IHC revealed low to weak NuMA expression in normal tissues. Expression was upregulated in tumours, with a significant association with disease stage in mucinous EOC subtypes (p = 0.009), lymph node involvement (p = 0.03) and patient age (p = 0.04). Additional discontinuous data analysis revealed that high NuMA levels in tumours decreased with grade (p = 0.02) but increased with disease stage (p = 0.04) in serous EOC. NuMA expression decreased in late disease stage 4 endometrioid EOCs. High NuMA levels decreased with increased tumour invasion in all subtypes (p = 0.03). IF of primary cultures revealed that high NuMA levels at mitotic spindle poles were significantly associated with a decreased proportion of cells in cytokinesis (p = 0.05), increased binucleation (p = 0.021) and multinucleation (p = 0.007), and aneuploidy (p = 0.008). NuMA is highly expressed in EOC tumours and high NuMA levels correlate with increases in mitotic defects and aneuploidy in primary cultures.

Most information about the roles of the adenomatous polyposis coli protein (APC) and its binding partner EB1 in mitotic cells has come from siRNA studies. These suggest functions in chromosomal segregation and spindle positioning whose loss might contribute to tumourigenesis in cancers initiated by APC mutation. However, siRNA-based approaches have drawbacks associated with the time taken to achieve significant expression knockdown and the pleiotropic effects of EB1 and APC gene knockdown. Here we describe the effects of microinjecting APC- or EB1- specific monoclonal antibodies and a dominant-negative EB1 protein fragment into mammalian mitotic cells. The phenotypes observed were consistent with the roles proposed for EB1 and APC in chromosomal segregation in previous work. However, EB1 antibody injection also revealed two novel mitotic phenotypes, anaphase-specific cortical blebbing and asymmetric spindle pole movement. The daughters of microinjected cells displayed inequalities in microtubule content, with the greatest differences seen in the products of mitoses that showed the severest asymmetry in spindle pole movement. Daughters that inherited the least mobile pole contained the fewest microtubules, consistent with a role for EB1 in processes that promote equality of astral microtubule function at both poles in a spindle. We propose that these novel phenotypes represent APC-independent roles for EB1 in spindle pole function and the regulation of cortical contractility in the later stages of mitosis. Our work confirms that EB1 and APC have important mitotic roles, the loss of which could contribute to CIN in colorectal tumour cells.

Background: The clinico-pathological and molecular heterogeneity of epithelial ovarian cancer (EOC) complicates its early diagnosis and successful treatment. Highly aneuploid tumours and the presence of ascitic fluids are hallmarks of EOC. Two microcephaly-associated proteins, abnormal spindle-like microcephaly-associated protein (ASPM) and microcephalin, are involved in mitosis and DNA damage repair. Their expression is deregulated at the RNA level in EOC. Here, ASPM and microcephalin protein expression in primary cultures established from the ascites of patients with EOC was determined and correlated with clinical data to assess their suitability as biomarkers. Methods: Five established ovarian cancer cell lines, cells derived from two benign ovarian ascites samples and 40 primary cultures of EOC derived from ovarian ascites samples were analysed by protein slot blotting and/or immunofluorescence to determine ASPM and microcephalin protein levels and their cellular localisation. Results were correlated with clinico-pathological data. Results: A statistically significant correlation was identified for ASPM localisation and tumour grade, with high levels of cytoplasmic ASPM correlating with grade 1 tumours. Conversely, cytoplasmic microcephalin was only identified in high-grade tumours. Furthermore, low levels of nuclear microcephalin correlated with reduced patient survival. Conclusion: Our results suggest that ASPM and microcephalin have the potential to be biomarkers in ovarian cancer.

The critical importance of cytoskeletal function for correct neuronal migration during development of the cerebral cortex has been underscored by the identities of germline mutations underlying a number of human neurodevelopmental disorders. The proteins affected include TUBA1A, a major alpha-tubulin isoform, and microtubule-associated components such as doublecortin, and LIS1. Mutations in these genes are associated with the anatomical abnormality lissencephaly, which is believed to reflect failure of neuronal migration. An important recent observation has been the dependence of cortical neuronal migration upon acetylation of alpha-tubulin at lysine 40 by the histone acetyltransferase Elongator complex. Here, we describe a recognizable autosomal recessive syndrome, characterized by generalized polymicrogyria in association with optic nerve hypoplasia (PMGOH). By autozygosity mapping, we show that the molecular basis for this condition is mutation of the TUBA8 gene, encoding a variant alpha-tubulin of unknown function that is not susceptible to the lysine 40 acetylation that regulates microtubule function during cortical neuron migration. Together with the unique expression pattern of TUBA8 within the developing cerebral cortex, these observations suggest a role for this atypical microtubule component in regulating mammalian brain development.

Treatment of an array of bis(phenol)s H2Lx {Lx = [(-OC6H2(2,4-R)(6-CH2))2 NCH2CH2X], where X = CH2NMe2, NMe2, NEt2, OMe and R = Bu(t), Pe(t) where Pe(t) = C(CH3)2Et} with [Ln(N(SiMe3)2)2(THF)2] (Ln = Yb, Sm) in a 1:1 molar ratio in hexanes afforded a multitude of new divalent lanthanide bis(phenolate) complexes: [L(2c)Yb]2 (X = NEt2, R = Bu(t)) 1, [L(2b)Yb]2 (X = CH2NMe2, R = Bu(t)) 2, [L(3a)Yb]2 (X = NMe2, R = Pe(t)) 3, [L(3b)Yb]2 (X = OMe, R = Pe(t)) 4, [L(2c)Sm] (X = NEt2, R = Bu(t)) 5, [L(3c)Yb] (X = NEt2, R = Pe(t)) 6, [L(3c)Sm] (X = NEt2, R = Pe(t)) 7. X-Ray crystallographic analyses of compounds 1 and 3 reveal dimeric, centrosymmetric structures with 5-coordinate ytterbium centers arising from bridging and terminal phenolate groups. A selection of divalent compounds (1, 3, 4, 5, 6 and 7) were tested as catalyst precursors in the polymerization of epsilon-caprolactone and/or L-lactide which resulted in high molecular weight polymers with PDIs of 1.11-2.81 and 1.13-1.56 for epsilon-caprolactone and L-lactide respectively. All polymerization studies were performed in either toluene or THF and at room temperature (epsilon-caprolactone) or 70 degrees C (L-lactide) at a variety of catalyst:monomer ratios (1:100-1:300). Kinetics analyses of the polymerization of L-lactide by compounds 1 and 5 indicated pseudo-first order response with respect to L-lactide.

More than 50% of transitional cell carcinomas of the bladder show loss of heterozygosity of a region spanning the TSC1 locus at 9q34 and mutations of TSC1 have been identified in 14.5% of tumours. These comprise nonsense mutations, splicing mutations, small deletions and missense mutations. Missense mutations are only rarely found in the germline in TSC disease. Therefore, we have examined six somatic missense mutations found in bladder cancer to determine whether these result in loss of function. We describe loss of function via distinct mechanisms. Five mutations caused mutually exclusive defects at mRNA and protein levels. Of these, two mutations caused pre-mRNA splicing errors that were predicted to result in premature protein truncation and three resulted in markedly reduced stability of exogenous TSC1 protein. Primary tumours with aberrant TSC1 pre-mRNA splicing were confirmed as negative for TSC1 expression by immunohistochemistry. Expression was also significantly reduced in a tumour with a TSC1 missense mutation resulting in diminished protein half-life. A single TSC1 missense mutation identified in a tumour with retained heterozygosity of the TSC1 region on chromosome 9 caused an apparently TSC2- and mTOR-independent localization defect of the mutant protein. We conclude that although TSC1 missense mutations do not play a major role in causation of TSC disease, they represent a significant proportion of somatic loss of function mutations in bladder cancer.

The microtubule (MT)-associated protein EB1 localizes to and promotes growth at MT plus ends. The MT depolymerizing kinesin MCAK has also been reported to track growing MT plus ends. Here, we confirm that human MCAK colocalizes with EB1 at growing MT ends when expressed as a GFP fusion protein in transfected cells. We show that MCAK associates with the C-terminus of EB1 and EB3 but much less efficiently with RP1. EB1 associates with the N-terminal localization and regulatory domain in MCAK but not with the motor domain of the protein. The interaction is competitive with the binding of other EB1 ligands and does not require MTs. Knockdown of EB1 expression using siRNA impaired the ability of GFP-MCAK to localize to MT tips in transfected cells. We propose that MCAK is targeted to growing MT ends by EB1, that MCAK is held in an inactive conformation when associated with EB1 and that this could provide the basis for a mechanism that facilitates rapid switching between phases of MT growth and depolymerization.

Five new heteroleptic lanthanide(III) phenolate compounds have been synthesised in high yield, four via a transamination reaction between Ln(N(SiMe(3))2)3 and two equivalents of the phenol, HOC(6)H(2)(2,4-Bu(t))-6-CH(2)N(Me)CH(2)CH(2)NMe(2) [corrected] (LH) in thf {L(2)LnN(SiMe(3))2 where Ln = La (1); Nd (2); Sm (3); Yb (4)}. The fifth compound, [L(2)La][BPh(4)] 5 was formed by conversion of 1 by treatment with one equivalent of [Et(3)NH][BPh(4)] in toluene. Compound 3 was subjected to a single-crystal X-ray analysis and revealed a five-coordinate, distorted trigonal bipyramidal samarium(III) metal centre where each phenolate ligand is bidentate coordinating through the phenolate oxygen and nitrogen yielding six-membered chelate rings. Compound 1 exhibited fluxional behaviour in C(4)D(8)O solution which was temperature dependent. All five compounds were assessed as catalyst precursors towards the ring-opening polymerisation of both L-lactide and epsilon-caprolactone. These polymerisation studies revealed that catalysts containing larger lanthanide metals were more efficacious than those with smaller lanthanide metals. Furthermore, replacement of the [N(SiMe(3))2] initiating group in 1 with [BPh(4)] in 5 reduced catalytic activity by this compound. Detailed kinetics analysis of the ring-opening polymerisation of L-lactide by compound 1, the most efficacious catalyst precursor analysed in this study, revealed the following rate law: -d[LA]/dt = k[LA](2)[1](1) which is second order in lactide and first order in catalyst. End-group analysis by ESI mass spectrometry revealed the presence of phenolate end-groups and lactide cycles, the latter formed by intra-molecular, intrachain transesterification.

Background The trafficking of the adenomatous polyposis coli (APC) tumour suppressor protein in mammalian cells is a perennially controversial topic. Immunostaining evidence for an actin-associated APC localisation at intercellular junctions has been previously presented, though live imaging of mammalian junctional APC has not been documented. Results Using live imaging of transfected COS-7 cells we observed intercellular junction-associated pools of GFP-APC in addition to previously documented microtubule-associated GFP-APC and a variety of minor localisations. Although both microtubule and junction-associated populations could co-exist within individual cells, they differed in their subcellular location, dynamic behaviour and sensitivity to cytoskeletal poisons. GFP-APC deletion mutant analysis indicated that a protein truncated immediately after the APC armadillo repeat domain retained the ability to localise to adhesive membranes in transfected cells. Supporting this, we also observed junctional APC immunostaining in cultures of human colorectal cancer cell line that express truncated forms of APC. Conclusion Our data indicate that APC can be found in two spatially separate populations at the cell periphery and these populations can co-exist in the same cell. The first localisation is highly dynamic and associated with microtubules near free edges and in cell vertices, while the second is comparatively static and is closely associated with actin at sites of cell-cell contact. Our imaging confirms that human GFP-APC possesses many of the localisations and behaviours previously seen by live imaging of Xenopus GFP-APC. However, we report the novel finding that GFP-APC puncta can remain associated with the ends of shrinking microtubules. Deletion analysis indicated that the N-terminal region of the APC protein mediated its junctional localisation, consistent with our observation that truncated APC proteins in colon cancer cell lines are still capable of localising to the cell cortex. This may have implications for the development of colorectal cancer.

Dendritic cells (DC) are professional antigen-presenting cells (APC) of the immune system, uniquely able to prime naive T-cell responses. They are the focus of a range of novel strategies for the immunotherapy of cancer, a proportion of which include treating DC with ionising radiation to high dose. The effects of radiation on DC have not, however, been fully characterised. We therefore cultured human myeloid DC from CD14+ precursors, and studied the effects of ionising radiation on their phenotype and function. Dendritic cells were remarkably resistant against radiation-induced apoptosis, showed limited changes in surface phenotype, and mostly maintained their endocytic, phagocytic and migratory capacity. However, irradiated DC were less effective in a mixed lymphocyte reaction, and on maturation produced significantly less IL-12 than unirradiated controls, while IL-10 secretion was maintained. Furthermore, peptide-pulsed irradiated mature DC were less effective at naive T-cell priming, stimulating fewer effector cells with lower cytotoxicity against antigen-specific targets. Hence irradiation of DC in vitro, and potentially in vivo, has a significant impact on their function, and may shift the balance between T-cell activation and tolerization in DC-mediated immune responses.

BACKGROUND Despite recent technical improvements, many human preimplantation embryos fail to develop to the blastocyst stage or implant after transfer to the uterus. A possible cause for this developmental arrest is the high incidence of nuclear and postzygotic chromosomal abnormalities observed during cleavage, including chaotic chromosome complements, suggestive of defects in mitotic chromosomal segregation. The underlying mechanisms are largely unknown, but similarities with chromosome instability in human cancers led to the proposal that cell cycle checkpoints may not operate at these early stages. METHODS To investigate this and to examine whether spindle abnormalities contribute to chromosome malsegregation, we have used fluorescence and confocal laser scanning microscopy, following immunolabelling with antibodies specific for alpha-tubulin, gamma-tubulin, or acetylated tubulin, combined with a DNA fluorochrome to visualize nuclei, spindle and chromosome configurations in normal and arrested human embryos, from cleavage to blastocyst stages. RESULTS In addition to frequent interphase nuclear abnormalities, we identify for the first time various spindle abnormalities including abnormal shape and chromosome loss and multipolar spindles at cleavage and blastocyst stages. CONCLUSIONS We propose that a major pathway leading to postzygotic chromosomal abnormalities is the formation of binucleate blastomeres with two centrosomes which result either in a bipolar spindle and division to two tetraploid blastomeres, or in a multipolar spindle, chromosome malsegregation and chromosomal chaos.

Renal cancer is one of the most chemoresistant tumor types. Using a panel of 10 established renal cancer cell lines that have not been subjected to prior drug selection, the range of functional resistance phenotypes to the tubulin-binding agents paclitaxel, vinblastine, vincristine and patupilone (epothilone B, EPO906) was determined, together with expression of P-glycoprotein (PgP), multidrug resistance associated protein-2 (MRP2) and major vault protein (MVP) proteins. The IC(50) values for vincristine correlated positively with PgP expression (r = 0.73; p = 0.031), with values for paclitaxel and vinblastine just failing to reach significance. A significant positive correlation was observed for sensitivity to paclitaxel and MRP2 expression only (r = 0.8; p = 0.013). MVP expression did not correlate with sensitivity to any of the drugs examined. All cell lines exhibited much greater sensitivity to patupilone, demonstrating for the first time the potential use of patupilone in this cancer. In tissue samples from chemotherapy-naive renal cell carcinoma (RCC) patients, marked downregulation or absence of PgP in many tumor cells with expression levels more similar to sensitive cell lines rather than the resistant lines was seen. Similarly, MRP2 was absent or only weakly present in tumor cells, whereas MVP was very strongly upregulated in most tumor samples. This study illustrating discrepancies between results exclusively based on studies in cell lines and findings in vivo suggests that the role of PgP and MRP2 in intrinsic resistance in RCC in vivo may be less than expected from the in vitro findings and supports a potential role for MVP on the basis of in vivo expression studies.

Background EB1 is a microtubule tip-associated protein that interacts with the APC tumour suppressor protein and the p150glued subunit of dynactin. We previously reported that an EB1 deletion mutant that retains both of these interactions but does not directly associate with microtubules (EB1-ΔN2-GFP) spontaneously formed perinuclear aggregates when expressed in COS-7 cells. Results In the present study live imaging indicated that EB1-ΔN2-GFP aggregates underwent dynamic microtubule-dependent changes in morphology and appeared to be internally cohesive. EB1-ΔN2-GFP aggregates were phase-dense structures that displayed microtubule-dependent accumulation around the centrosome, were immunoreactive for both the 20s subunit of the proteasome and ubiquitin, and induced the collapse of the vimentin cytoskeleton. Fractionation studies revealed that a proportion of EB1-ΔN2-GFP was detergent-insoluble and ubiquitylated, indicating that EB1-ΔN2-GFP aggregates are aggresomes. Immunostaining also revealed that APC and p150glued were present in EB1-ΔN2-GFP aggregates, whereas EB3 was not. Furthermore, evidence for p150glued degradation was found in the insoluble fraction of EB1-ΔN2-GFP transfected cultures. Conclusion Our data indicate that aggresomes can be internally cohesive and may not represent a simple "aggregate of aggregates" assembled around the centrosome. Our observations also indicate that a partially misfolded protein may retain the ability to interact with its normal physiological ligands, leading to their co-assembly into aggresomes. This supports the idea that the trapping and degradation of co-aggregated proteins might contribute to human pathologies characterised by aggresome formation.

During mitosis, the spindle checkpoint delays the onset of anaphase until all chromosomes have attached properly to the mitotic spindle, preventing chromosome missegregation. BUB (budding uninhibited by benzimidazole) 1 is one of the key components of this checkpoint. BUB1 mutations are rare in cancer tissues and no mutations have been identified in gastric cancer. In mice, immunodepletion of BUB1 abolished the spindle checkpoint. Thus, aberrant expression of BUB1 protein could impair mitotic checkpoint function, resulting in aneuploidy, a common phenomenon in gastric cancer. In the present study, an antibody was generated against BUB1 and its expression was studied in gastric cancer tissue sections (n = 80) by immunohistochemistry. Nuclear BUB1 expression was found in all gastric cancer cases. The proportion of tumour cells expressing BUB1 was significantly greater in diffuse-type than in intestinal-type gastric carcinoma (p0.001). No correlation was found between BUB1 expression and deoxyribonucleic acid (DNA) ploidy, microsatellite instability or any other histopathological parameters investigated. To the authors' knowledge, this is the first study of BUB1 protein expression in gastric cancer tissues. Different BUB1 protein expression levels in intestinal- and diffuse-type gastric cancer may provide further evidence of a potential link between different genetic pathways and morphological phenotype in gastric carcinogenesis. However, further studies are needed to establish whether there is an association between BUB1 protein expression level and mitotic spindle checkpoint function in gastric cancer.

CLIP-170 is a “cytoplasmic linker protein” implicated in endosome-microtubule interactions and in control of microtubule dynamics. CLIP-170 localizes dynamically to growing microtubule plus ends, colocalizing with the dynein activator dynactin and the APC-binding protein EB1. This shared “plus-end tracking” behavior suggests that CLIP-170 might interact with dynactin and/or EB1. We have used site-specific mutagenesis of CLIP-170 and a transfection/colocalization assay to address this question in mammalian tissue culture cells. Our results indicate that CLIP-170 interacts, directly or indirectly, with both dynactin and EB1. We find that the CLIP-170/dynactin interaction is mediated by the second metal binding motif of the CLIP-170 tail. In contrast, the CLIP-170/EB1 interaction requires neither metal binding motif. In addition, our experiments suggest that the CLIP-170 /dynactin interaction occurs via the shoulder/sidearm subcomplex of dynactin and can occur in the cytosol (i.e., it does not require microtubule binding). These results have implications for the targeting of both dynactin and EB1 to microtubule plus ends. Our data suggest that the CLIP-170/dynactin interaction can target dynactin complex to microtubule plus ends, although dynactin likely also targets MT plus ends directly via the microtubule binding motif of the p150Glued subunit. We find that CLIP-170 mutants alter p150Glued localization without affecting EB1, indicating that EB1 can target microtubule plus ends independently of dynactin. Cell Motil. Cytoskeleton 55:156–173, 2003. © 2003 Wiley-Liss, Inc.

Objective The current treatment of hydrocephalus using ventriculoperitoneal shunts and third ventriculostomies remains problematic. We revisited the concept of destruction of the choroid plexus for the treatment of hydrocephalus by using an immunotoxin-based technique to specifically destroy this tissue. This approach was based on the observation that, as an epithelial tissue, choroid plexus expresses a number of specific cell-surface proteins that represent excellent potential targets for the creation of a choroid plexus-specific immunotoxin. Methods In this study, we characterized sheep and human choroid plexus cells (including atypical and carcinoma cell lines) using fluorescence microscopy in combination with histochemical staining of rat brain and confirmed the presence of a number of epithelium-specific proteins in choroid plexus cells. Immunotoxins were then manufactured by linking these antibodies to ricin A chain and ricin A-B chain. These immunotoxins were delivered to choroid plexus-derived cells in culture, and the results were compared with results of exposure to a nonspecific immunotoxin. Results Complete cell death of choroid plexus cells was seen after only a 1-hour exposure to the specific immunotoxin, as opposed to the minimal cell death seen with a nonspecific immunotoxin after several hours of exposure. Conclusion These results suggest that immunotoxin-mediated ablation of choroid plexus may be a viable method of treating hydrocephalus and choroid plexus-derived tumors.