bluuebunny/crossref_metadata_2025 huggingface dataset:
Measurements of spectral similarity for microearthquakes in western Nagano, Japan
Authors: Venkataraman Anupama , Beroza Gregory C. , Ide Satoshi , Imanishi Kazutoshi , Ito Hisao , Iio Yoshihisa
Published: March 2006

We use P wave spectral ratios to estimate the seismic energy of 23 microearthquakes (Mw ≈ 0.9–2.7) recorded on a dense network of surface and borehole stations in western Nagano, Japan. These events were recorded at 45 surface stations and 2 borehole stations. The data set is unique in that most events have high signal‐to‐noise ratio at several surface stations and at the two borehole stations. The redundancy provided by the large number of stations recording these events allows us to assess the uncertainty of our results. We find that the seismic energy to moment (ER/M0) ratios from P waves for the events in our data set are reliable, that they vary between 1 × 10−6 and 1 × 10−4, and that smaller events tend to have smaller values of ER/M0 because they are relatively deficient in radiated energy above the corner frequency. We observe two spectral effects: a variation in corner frequency, which accounts for most of the variation in ER/M0, and steeper spectral falloff rates, which contribute to the small values of ER/M0 for some of the smaller events. Our estimates of moment and corner frequency suggest that M0 ∝ fc−(3+e) (ɛ > 0), and this implies that either the static stress drop or rupture velocity or both change with earthquake size.
Incorporation of partial polyhedrin homology sequences (PPHS) enhances the production of cloned foreign genes in a baculovirus expression system
Authors: Gong Zhaohui , Jin Yongfeng , Zhang Yaozhou
Published: March 2006

Baculovirus expression vector systems (BEVSs) have been used extensively for high‐level expression of cloned foreign genes. In many instances, the levels of recombinant protein(s) produced in insect cells and larvae are insufficient for experimental purposes. Thus new techniques and methods are needed to increase significantly the protein expression levels in BEVS. In the present paper, we describe the incorporation of a 15 bp element derived from the 5′‐end partial sequence of the polyhedrin gene, which contains the non‐coding sequence ATAAAT and the coding sequence ATGCCGAAT, into the 5′‐end of the CTB (cholera toxin B subunit)–INS (insulin) fusion gene. With the addition of the PPHS (partial polyhedrin homology sequences), two extra amino acids (Pro‐Asn) were added to the N‐terminus of the mCTB–INS (modified CTB–INS) fusion protein. This new fusion protein was expressed in both insect cells and larvae using BEVSs. We found that the addition of PPHS enhanced 4‐fold the expression of CTB–INS in both insect cells and larvae. Further analysis revealed that the additional two amino acids in mCTB–INS did not significantly affect binding affinity for GM1 ganglioside. Therefore the PPHS can be used as a constitutive element immediately downstream of the polyhedrin promoter to induce significant increases in the expression levels of cloned foreign genes.
The transcription factors Slug and Snail act as repressors of Claudin-1 expression in epithelial cells
Authors: Martínez-Estrada Ofelia M. , Cullerés Albert , Soriano Francesc X. , Peinado Hector , Bolós Victoria , Martínez Fernando O. , Reina Manuel , Cano Amparo , Fabre Myriam , Vilaró Senén
Published: March 2006

Claudin-1 is an integral membrane protein component of tight junctions. The Snail family of transcription factors are repressors that play a central role in the epithelial–mesenchymal transition, a process that occurs during cancer progression. Snail and Slug members are direct repressors of E-cadherin and act by binding to the specific E-boxes of its proximal promoter. In the present study, we demonstrate that overexpression of Slug or Snail causes a decrease in transepithelial electrical resistance. Overexpression of Slug and Snail in MDCK (Madin–Darby canine kidney) cells down-regulated Claudin-1 at protein and mRNA levels. In addition, Snail and Slug are able to effectively repress human Claudin-1-driven reporter gene constructs containing the wild-type promoter sequence, but not those with mutations in two proximal E-box elements. We also demonstrate by band-shift assay that Snail and Slug bind to the E-box motifs present in the human Claudin-1 promoter. Moreover, an inverse correlation in the levels of Claudin-1 and Slug transcripts were observed in breast cancer cell lines. E-box elements in the Claudin-1 promoter were found to play a critical negative regulatory role in breast cancer cell lines that expressed low levels of Claudin-1 transcript. Significantly, in invasive human breast tumours, high levels of Snail and Slug correlated with low levels of Claudin-1 expression. Taken together, these results support the hypothesis that Claudin-1 is a direct downstream target gene of Snail family factors in epithelial cells.
The N-terminal domain of the human eIF2β subunit and the CK2 phosphorylation sites are required for its function
Authors: Llorens Franc , Duarri Anna , Sarró Eduard , Roher Nerea , Plana Maria , Itarte Emilio
Published: March 2006

CK2 (protein kinase CK2) is known to phosphorylate eIF2 (eukaryotic translation initiation factor 2) in vitro; however, its implication in this process in living cells has remained to be confirmed. The combined use of chemical inhibitors (emodin and apigenin) of CK2 together with transfection experiments with the wild-type of the K68A kinase-dead mutant form of CK2α evidenced the direct involvement of this protein kinase in eIF2β phosphorylation in cultured HeLa cells. Transfection of HeLa cells with human wild-type eIF2β or its phosphorylation site mutants showed Ser2 as the main site for constitutive eIF2β phosphorylation, whereas phosphorylation at Ser67 seems more restricted. In vitro phosphorylation of eIF2β also pointed to Ser2 as a preferred site for CK2 phosphorylation. Overexpression of the eIF2β S2/67A mutant slowed down the rate of protein synthesis stimulated by serum, although less markedly than the overexpression of the Δ2–138 N-terminal-truncated form of eIF2β (eIF2β-CT). Mutation at Ser2 and Ser67 did not affect eIF2β integrating into the eIF2 trimer or being able to complex with eIF5 and CK2α. The eIF2β-CT form was also incorporated into the eIF2 trimer but did not bind to eIF5. Overexpression of eIF2β slightly decreased HeLa cell viability, an effect that was more evident when overexpressing the eIF2β S2/67A mutant. Cell death was particularly marked when overexpressing the eIF2β-CT form, being detectable at doses where eIF2β and eIF2β S2/67A were ineffective. These results suggest that Ser2 and Ser67 contribute to the important role of the N-terminal region of eIF2β for its function in mammals.
Cell binding, internalization and cytotoxic activity of human granzyme B expressed in the yeastPichia pastoris
Authors: Giesübel Ulrike , Dälken Benjamin , Mahmud Hayat , Wels Winfried S.
Published: March 2006

Granzyme B (GrB) is an apoptosis-inducing protease of cytotoxic lymphocytes. We have investigated intracellular and extracellular effects of human GrB using recombinant protein expressed in the yeast Pichia pastoris. GrB was rapidly taken up by HeLa cells, and accumulated in vesicular structures in the cytoplasm. There it remained inactive and could not be liberated by the endosomolytic reagent chloroquine, indicating that the vesicular structures are distinct from late endosomes and lysosomes. Direct cytosolic delivery of GrB with a cationic lipid-based transduction reagent, however, resulted in the induction of apoptotic cell death. After prolonged incubation at or above 125 nM, GrB on its own induced pronounced morphological changes in human tumour cells, leading to partial loss of contact to the culture support. This extracellular effect was dependent on enzymatic activity and could be reversed by removal of the protein, suggesting GrB-dependent cleavage of extracellular matrix components as the underlying mechanism.
Salivary agglutinin and lung scavenger receptor cysteine-rich glycoprotein 340 have broad anti-influenza activities and interactions with surfactant protein D that vary according to donor source and sialylation
Authors: Hartshorn Kevan L. , Ligtenberg Antoon , White Mitchell R. , van Eijk Martin , Hartshorn Max , Pemberton Lily , Holmskov Uffe , Crouch Erika
Published: March 2006

We previously found that scavenger receptor cysteine-rich gp-340 (glycoprotein-340), isolated from lung or saliva, directly inhibits human IAVs (influenza A viruses). We now show that salivary gp-340 has broad antiviral activity against human, equine and porcine IAV strains. Although lung and salivary gp-340 are identical in protein sequence, salivary gp-340 from one donor had significantly greater antiviral activity against avian-like IAV strains which preferentially bind sialic acids in α(2,3) linkage. A greater density of α(2,3)-linked sialic acids was present on the salivary gp-340 from this donor as compared with salivary gp-340 from another donor or several preparations of lung gp-340. Hence, the specificity of sialic acid linkages on gp-340 is an important determinant of anti-IAV activity. Gp-340 binds to SP-D (surfactant protein D), and we previously showed that lung gp-340 has co-operative interactions with SP-D in viral neutralization and aggregation assays. We now report that salivary gp-340 can, in some cases, strongly antagonize certain antiviral activities of SP-D. This effect was associated with greater binding of salivary gp-340 to the carbohydrate recognition domain of SP-D as compared with the binding of lung gp-340. These findings may relate to inter-individual variations in innate defence against highly pathogenic IAV and to effects of aspiration of oral contents on SP-D-mediated lung functions.
Thioredoxin reductase regulates the induction of haem oxygenase-1 expression in aortic endothelial cells
Authors: Trigona Wendy L. , Mullarky Isis K. , Cao Yuzhang , Sordillo Lorraine M.
Published: March 2006

Certain selenoproteins such as GPX-1 (glutathione peroxidase-1) and TrxR1 (thioredoxin reductase-1) possess important antioxidant defence functions in vascular endothelial cells. Reduced selenoprotein activity during dietary selenium (Se) deficiency can result in a compensatory increase of other non-Se-dependent antioxidants, such as HO-1 (haem oxygenase-1) that may help to counteract the damaging effects of oxidant stress. However, the role of individual selenoproteins in regulating vascular-derived protective gene responses such as HO-1 is less understood. Using an oxidant stress model based on Se deficiency in BAECs (bovine aortic endothelial cells), we sought to determine whether TrxR1 activity may contribute to the differential regulation of HO-1 expression as a function of altered redox environment. Se-sufficient BAECs up-regulated HO-1 expression following stimulation with the pro-oxidant, 15-HPETE (15-hydroperoxyeicosatetraenoic acid), and levels of this antioxidant inversely correlated with EC apoptosis. While Se-deficient BAECs exhibited higher basal levels of HO-1, it was not up-regulated upon 15-HPETE treatment, which resulted in significantly higher levels of pro-apoptotic markers. Subsequent results showed that HO-1 induction depended on the activity of TrxR1, as proved with chemical inhibitor studies and direct inhibition with TrxR1 siRNA. Finally, restoring intracellular levels of the reduced substrate Trx (thioredoxin) in Sedeficient BAECs was sufficient to increase HO-1 activation following 15-HPETE stimulation. These data provide evidence for the involvement of the Trx/TrxR system, in the regulation of HO-1 expression in BAECs during pro-oxidant challenge.
Vasodilator-stimulated phosphoprotein (VASP) is phosphorylated on Ser157 by protein kinase C-dependent and -independent mechanisms in thrombin-stimulated human platelets
Authors: Wentworth James K. T. , Pula Giordano , Poole Alastair W.
Published: March 2006

VASP (vasodilator-stimulated phosphoprotein) is an actin- and profilin-binding protein that is expressed in platelets at high levels and plays a major role in negatively regulating secretory and adhesive events in these cells. VASP is a major substrate for cAMP- and cGMP-regulated protein kinases and it has been shown to be directly phosphorylated on Ser157 by PKC (protein kinase C). In the present paper, we show that, in human platelets, VASP is phosphorylated by PKC on Ser157, but not Ser239, in response to phorbol ester stimulation, in a manner blocked by the PKC inhibitor BIM I (bisindolylmaleimide I). In response to thrombin, VASP was also phosphorylated on Ser157, but this response was only partially inhibited by BIM I, indicating PKC-dependent and -independent pathways to VASP phosphorylation by thrombin. Using inhibitors, we have ruled out the possibility that the PKC-independent pathway acts through guanylate cyclase generation of cGMP, or through a phosphoinositide 3-kinase-dependent kinase. Inhibition of Rho kinase, however, substantially reduced Ser157 VASP phosphorylation, and its effects were additive with BIM I. This implicates Rho kinase and PKC as the major kinases that phosphorylate VASP Ser157 in response to thrombin in platelets.
Kinetic, inhibition and structural studies on 3-oxoacyl-ACP reductase from Plasmodium falciparum, a key enzyme in fatty acid biosynthesis
Authors: Wickramasinghe Sasala R. , Inglis Kirstine A. , Urch Jonathan E. , Müller Sylke , van Aalten Daan M. F. , Fairlamb Alan H.
Published: March 2006

Type II fatty acid biosynthesis represents an attractive target for the discovery of new antimalarial drugs. Previous studies have identified malarial ENR (enoyl acyl-carrier-protein reductase, or FabI) as the target for the antiseptic triclosan. In the present paper, we report the biochemical properties and 1.5 Å (1 Å=0.1 nm) crystal structure of OAR (3-oxoacyl acyl-carrier-protein reductase, or FabG), the second reductive step in fatty acid biosynthesis and its inhibition by hexachlorophene. Under optimal conditions of pH and ionic strength, Plasmodium falciparum OAR displays kinetic properties similar to those of OAR from bacteria or plants. Activity with NADH is <3% of that with NADPH. Fluorescence enhancement studies indicate that NADPH can bind to the free enzyme, consistent with kinetic and product inhibition studies suggesting a steady-state ordered mechanism. The crystal structure reveals a tetramer with a sulphate ion bound in the cofactor-binding site such that the side chains of the catalytic triad of serine, tyrosine and lysine are aligned in an active conformation, as previously observed in the Escherichia coli OAR–NADP+ complex. A cluster of positively charged residues is positioned at the entrance to the active site, consistent with the proposed recognition site for the physiological substrate (3-oxoacyl-acyl-carrier protein) in E. coli OAR. The antibacterial and anthelminthic agent hexachlorophene is a potent inhibitor of OAR (IC50 2.05 μM) displaying non-linear competitive inhibition with respect to NADPH. Hexachlorophene (EC50 6.2 μM) and analogues such as bithionol also have antimalarial activity in vitro, suggesting they might be useful leads for further development.
Effects of N-linked glycosylation on the creatine transporter
Authors: Straumann Nadine , Wind Alexandra , Leuenberger Tina , Wallimann Theo
Published: March 2006

The CRT (creatine transporter) is a member of the Na+- and Cl−-dependent neurotransmitter transporter family and is responsible for the import of creatine into cells, and thus is important for cellular energy metabolism. We established for CRT an expression system in HEK-293 cells that allowed biochemical, immunological and functional analysis of CRT wild-type and glycosylation-deficient mutants. Analysis of HA (haemagglutinin)-tagged CRT-NN (wild-type rat CRT with an HA-tag at the C-terminus) revealed several monomeric immunoreactive species with apparent molecular masses of 58, 48 and 43 kDa. The 58 kDa species was shown to be plasma-membrane-resident by EndoHf (endoglycosidase Hf) and PNGase F (peptide N-glycosidase F) treatments and represents fully glycosylated CRT, whereas the 48 kDa and 43 kDa species were glycosylation intermediates and non-glycosylated CRT respectively. Glycosylation-deficient mutants (Asn192Asp, Asn197Asp and Asn192Asp/Asn197Asp) showed altered electrophoretic mobility, indicating that CRT is indeed N-glycosylated. In addition, a prominent CRT band in the range of 75–91 kDa was also detected. Pharmacological inhibition of N-linked glycosylation by tunicamycin in CRT-NN-expressing cells gave a similar reduction in molecular mass, corroborating the finding that Asn192 and Asn197 are major N-glycosylation sites in CRT. Although the apparent Km was not significantly affected in glycosylation-deficient mutants compared with CRT-NN, we measured reduced Vmax values for all mutants (21–28% residual activity), and 51% residual activity after enzymatic deglycosylation of surface proteins in intact CRT-NN cells by PNGase F. Moreover, immunocytochemical analysis of CRT-NN- and CRT-DD-expressing cells (where CRT-DD represents a non-glycosylated double mutant of CRT, i.e. Asn192Asp/Asn197Asp) showed a lower abundance of CRT-DD in the plasma membrane. Taken together, our results suggest that plasma-membrane CRT is glycosylated and has an apparent monomer molecular mass of 58 kDa. Furthermore, N-linked glycosylation is neither exclusively important for the function of CRT nor for surface trafficking, but affects both processes. These findings may have relevance for closely related neurotransmitter transporter family members.
Reactivity of free thiol groups in type-I inositol trisphosphate receptors
Authors: Joseph Suresh K. , Nakao Steven K. , Sukumvanich Siam
Published: March 2006

The IP3R (inositol 1,4,5-trisphosphate receptor) Ca2+-release channel is known to be sensitive to thiol redox state. The present study was undertaken to characterize the number and location of reactive thiol groups in the type-I IP3R. Using the fluorescent thiol-reactive compound monobromobimane we found that approx. 70% of the 60 cysteine residues in the type-I IP3R are maintained in the reduced state. The accessibility of these residues was assessed by covalently tagging the IP3R in membranes with a 5 kDa or 20 kDa MPEG [methoxypoly(ethylene glycol) maleimide]. MPEG reaction caused a shift in the mobility of IP3R on SDS/PAGE that was blocked by pretreatment of the membranes with dithiothreitol, N-ethylmaleimide, mersalyl or thimerosal, indicating that MPEG reactivity was specific to thiol groups on the IP3R. Trypsin cleavage of the type-I IP3R generates five defined domains. In cerebellum membranes, MPEG reacted over a 5 min interval with tryptic fragment I and fragment III, but not fragments II, IV or V. Fragment I appears as a doublet in cerebellum membranes, corresponding to the presence and absence of the SI splice site in this region (SI is a spliced domain corresponding to amino acids 318–332). Only the fragment I band corresponding to the SI(+) splice form shifted after reaction with MPEG. Expression of SI(+) and SI(−) spliced forms in COS cell microsomes confirmed this result. The MPEG-induced shift was not prevented when the cysteine residue present in the SI splice domain (C326A) or the remaining seven cysteine residues in fragment I were individually mutated. Of the combination mutations screened, only the mutation of C206/214/326A blocked MPEG reactivity in fragment I. We conclude that a set of highly reactive cysteine residues in fragment I are differentially accessible in the SI(+) and SI(−) splice variants of the type-I IP3R.
Inhibition of GLI1 gene activation by Patched1
Authors: Rahnama Fahimeh , Shimokawa Takashi , Lauth Matthias , Finta Csaba , Kogerman Priit , Teglund Stephan , Toftgård Rune , Zaphiropoulos Peter G.
Published: March 2006

Patched1 (PTCH1) is a human tumour suppressor that acts as an HH (Hedgehog) receptor protein and is important for embryonic patterning. PTCH1 mediates its effects through SMO (Smoothened) and represses the expression of HH target genes such as the transcription factor GLI1 (glioma 1) as well as PTCH1. Up-regulation of these genes has been observed in several cancer forms, including basal cell carcinoma, digestive track tumours and small cell lung cancer. The fact that PTCH1 down-regulates its own expression via ‘negative feedback’ is an important feature in HH signalling, as it keeps the balance between HH and PTCH1 activities that are essential for normal development. In the present study, we provide evidence that a novel mechanism allowing PTCH1 to maintain this balance may also exist. We show that gene activation by GLI1, the transcriptional effector of the pathway, can be down-regulated by PTCH1 without involvement of the canonical cascade of HH signalling events. Specifically, the SMO antagonist cyclopamine has no appreciable effects in blocking this PTCH1-mediated inhibition. Moreover, the negative GLI1 regulator SUFU (Suppressor of Fused) was also found to be dispensable. Additionally, deletion mapping of PTCH1 has revealed that the domains encompassed by amino acids 180–786 and 1058–1210 are of highest significance in inhibiting GLI1 gene activation. This contrasts with the importance of the PTCH1 C-terminal domain for HH signalling.
A novel ARF-binding protein (LZAP) alters ARF regulation of HDM2
Authors: Wang Jialiang , He Xiaping , Luo Ying , Yarbrough Wendell G.
Published: March 2006

The tumour suppressor ARF (alternative reading frame) is encoded by the INK4a (inhibitor of cyclin-dependent kinase 4)/ARF locus, which is frequently altered in human tumours. ARF binds MDM2 (murine double minute 2) and releases p53 from inhibition by MDM2, resulting in stabilization, accumulation and activation of p53. Recently, ARF has been found to associate with other proteins, but, to date, little is known about ARF-associated proteins that are implicated in post-translational regulation of ARF activity. Using a yeast two-hybrid screen, we have identified a novel protein, LZAP (LXXLL/leucine-zipper-containing ARF-binding protein), that interacts with endogenous ARF in mammalian cells. In the present study, we show that LZAP reversed the ability of ARF to inhibit HDM2's ubiquitin ligase activity towards p53, but simultaneously co-operated with ARF, maintaining p53 stability and increasing p53 transcriptional activity. Expression of LZAP, in addition to ARF, increased the percentage of cells in the G1 phase of the cell cycle. Expression of LZAP also caused activation of p53 and a p53-dependent G1 cell-cycle arrest in the absence of ARF. Taken together, our data suggest that LZAP can regulate ARF biochemical and biological activity. Additionally, LZAP has p53-dependent cell-cycle effects that are independent of ARF.
Comparative characterization and expression analysis of the four Old Yellow Enzyme homologues from Shewanella oneidensis indicate differences in physiological function
Authors: Brigé Ann , Van Den Hemel Debbie , Carpentier Wesley , De Smet Lina , Van Beeumen Jozef J.
Published: March 2006

Shewanella oneidensis contains four genes that encode proteins that have high sequence identity with yeast OYE (Old Yellow Enzyme, an NADPH oxidoreductase), the well-studied archetype of the OYE protein family. The present paper describes the first comparative study of OYEs that are present in a single bacterial species, performed to gain insight into their biochemical properties and physiological importance. The four proteins [named SYE1–SYE4 (Shewanella Yellow Enzyme 1–4)] were expressed as glutathione S-transferase fusion proteins in Escherichia coli. The yield of SYE2, however, was too low for further characterization, even after expression attempts in S. oneidensis. The SYE1, SYE3 and SYE4 proteins were found to have characteristics similar to those of other OYE family members. They were identified as flavoproteins that catalyse the reduction of different α,β-unsaturated carbonyl compounds and form charge transfer complexes with a range of phenolic compounds. Whereas the properties of SYE1 and SYE3 were very similar, those of SYE4 were clearly different in terms of ligand binding, catalytic efficiency and substrate specificity. Also, the activity of SYE4 was found to be NADPH-dependent, whereas SYE1 and SYE3 had a preference for NADH. It has been suggested that yeast OYE protects the actin cytoskeleton from oxidative stress. There are indications that bacterial OYEs are also involved in the oxidative stress response, but their exact role is unclear. Induction studies in S. oneidensis revealed that yeast and bacterial OYEs may share a common physiological role, i.e. the protection of cellular components against oxidative damage. As only SYE4 was induced under oxidative stress conditions, however, a functional divergence between bacterial OYEs is likely to exist.
The differential role of Smad2 and Smad3 in the regulation of pro-fibrotic TGFβ1 responses in human proximal-tubule epithelial cells
Authors: Phanish Mysore K. , Wahab Nadia A. , Colville-Nash Paul , Hendry Bruce M. , Dockrell Mark E. C.
Published: March 2006

In chronic renal diseases, progressive loss of renal function correlates with advancing tubulo-interstitial fibrosis. TGFβ1-Smad (transforming growth factor-β1–Sma and Mad protein) signalling plays an important role in the development of renal tubulo-interstitial fibrosis. Secretion of CTGF (connective-tissue growth factor; CCN2) by PTECs (proximal-tubule epithelial cells) and EMT (epithelial–mesenchymal transdifferentiation) of PTECs to myofibroblasts in response to TGFβ are critical Smad-dependent events in the development of tubulo-interstitial fibrosis. In the present study we have investigated the distinct contributions of Smad2 and Smad3 to expression of CTGF, E-cadherin, α-SMA (α-smooth-muscle actin) and MMP-2 (matrix-metalloproteinase-2) in response to TGFβ1 treatment in an in vitro culture model of HKC-8 (transformed human PTECs). RNA interference was used to achieve selective and specific knockdown of Smad2 and Smad3. Cellular E-cadherin, α-SMA as well as secreted CTGF and MMP-2 were assessed by Western immunoblotting. TGFβ1 treatment induced a fibrotic phenotype with increased expression of CTGF, MMP-2 and α-SMA, and decreased expression of E-cadherin. TGFβ1-induced increases in CTGF and decreases in E-cadherin expression were Smad3-dependent, whereas increases in MMP-2 expression were Smad2-dependent. Increases in α-SMA expression were dependent on both Smad2 and Smad3 and were abolished by combined knockdown of both Smad2 and Smad3. In conclusion, we have demonstrated distinct roles for Smad2 and Smad3 in TGFβ1-induced CTGF expression and markers of EMT in human PTECs. This can be of therapeutic value in designing targeted anti-fibrotic therapies for tubulo-interstitial fibrosis.
Semaphorin 4D activates the MAPK pathway downstream of plexin-B1
Authors: Aurandt Jennifer , Li Weiquan , Guan Kun-Liang
Published: March 2006

Semaphorins are a large family of transmembrane and secreted proteins that signal primarily through the receptor plexin. Semaphorins have been characterized in the nervous system as axon guidance cues; however, they have also been shown to control development of other cellular systems such as the vasculature and lungs. As the role of semaphorins outside of the nervous system has broadened, so has elucidation of the intracellular signalling pathways they initiate. Previously, we and others have shown that plexin-B1 activates RhoA through the binding and activation of RhoGEF (guanine nucleotide-exchange factor)/LARG (leukaemia-associated RhoGEF) in response to semaphorin 4D stimulation. In the present study, we show that semaphorin 4D activates the MAPK (mitogen-activated protein kinase) pathway. We have found that the mechanism of activation requires the C-terminus of plexin-B1 and the activation of RhoA.
Agonist-evoked inositol trisphosphate receptor (IP3R) clustering is not dependent on changes in the structure of the endoplasmic reticulum
Authors: Chalmers Mark , Schell Michael J. , Thorn Peter
Published: March 2006

The size and number of IP3R (inositol 1,4,5-trisphosphate receptor) clusters located on the surface of the ER (endoplasmic reticulum) is hypothesized to regulate the propagation of Ca2+ waves in cells, but the mechanisms by which the receptors cluster are not understood. Using immunocytochemistry, live-cell imaging and heterologous expression of ER membrane proteins we have investigated IP3R clustering in the basophilic cell line RBL-2H3 following the activation of native cell-surface antigen receptors. IP3R clusters are present in resting cells, and upon receptor stimulation, form larger aggregates. Cluster formation and maintenance required the presence of extracellular Ca2+ in both resting and stimulated cells. Using transfection with a marker of the ER, we found that the ER itself also showed structural changes, leading to an increased number of ‘hotspots’, following antigen stimulation. Surprisingly, however, when we compared the ER hotspots and IP3R clusters, we found them to be distinct. Imaging of YFP (yellow fluorescent protein)–IP3R transfected in to living cells confirmed that IP3R clustering increased upon stimulation. Photobleaching experiments showed that the IP3R occupied a single contiguous ER compartment both before and after stimulation, suggesting a dynamic exchange of IP3R molecules between the clusters and the surrounding ER membrane. It also showed a decrease in the mobile fraction after cell activation, consistent with receptor anchoring within clusters. We conclude that IP3R clustering in RBL-2H3 cells is not simply a reflection of bulk-changes in ER structure, but rather is due to the receptor undergoing homotypic or heterotypic protein–protein interactions in response to agonist stimulation.
Metallothionein protects against oxidative stress-induced lysosomal destabilization
Authors: Baird Sarah K. , Kurz Tino , Brunk Ulf T.
Published: March 2006

The introduction of apo-ferritin or the iron chelator DFO (desferrioxamine) conjugated to starch into the lysosomal compartment protects cells against oxidative stress, lysosomal rupture and ensuing apoptosis/necrosis by binding intralysosomal redox-active iron, thus preventing Fenton-type reactions and ensuing peroxidation of lysosomal membranes. Because up-regulation of MTs (metallothioneins) also generates enhanced cellular resistance to oxidative stress, including X-irradiation, and MTs were found to be capable of iron binding in an acidic and reducing lysosomal-like environment, we propose that these proteins might similarly stabilize lysosomes following autophagocytotic delivery to the lysosomal compartment. Here, we report that Zn-mediated MT up-regulation, assayed by Western blotting and immunocytochemistry, results in lysosomal stabilization and decreased apoptosis following oxidative stress, similar to the protection afforded by fluid-phase endocytosis of apo-ferritin or DFO. In contrast, the endocytotic uptake of an iron phosphate complex destabilized lysosomes against oxidative stress, but this was suppressed in cells with up-regulated MT. It is suggested that the resistance against oxidative stress, known to occur in MT-rich cells, may be a consequence of autophagic turnover of MT, resulting in reduced iron-catalysed intralysosomal peroxidative reactions.
Gangliosides of myelosupportive stroma cells are transferred to myeloid progenitors and are required for their survival and proliferation
Authors: Ziulkoski Ana L. , Andrade Cláudia M. B. , Crespo Pilar M. , Sisti Elisa , Trindade Vera M. T. , Daniotti Jose L. , Guma Fátima C. R. , Borojevic Radovan
Published: March 2006

In previous studies, we have shown that the myelopoiesis dependent upon myelosupportive stroma required production of growth factors and heparan-sulphate proteoglycans, as well as generation of a negatively charged sialidase-sensitive intercellular environment between the stroma and the myeloid progenitors. In the present study, we have investigated the production, distribution and role of gangliosides in an experimental model of in vitro myelopoiesis dependent upon AFT-024 murine liver-derived stroma. We used the FDC-P1 cell line, which is dependent upon GM-CSF (granulocyte/macrophage colony-stimulating factor) for both survival and proliferation, as a reporter system to monitor bioavailability and local activity of GM-CSF. GM3 was the major ganglioside produced by stroma, but not by myeloid cells, and it was required for optimal stroma myelosupportive function. It was released into the supernatant and selectively incorporated into the myeloid progenitor cells, where it segregated into rafts in which it co-localized with the GM-CSF-receptor α chain. This ganglioside was also metabolized further by myeloid cells into gangliosides of the a and b series, similar to endogenous GM3. In these cells, GM1 was the major ganglioside and it was segregated at the interface by stroma and myeloid cells, partially co-localizing with the GM-CSF-receptor α chain. We conclude that myelosupportive stroma cells produce and secrete the required growth factors, the cofactors such as heparan sulphate proteoglycans, and also supply gangliosides that are transferred from stroma to target cells, generating on the latter ones specific membrane domains with molecular complexes that include growth factor receptors.
Catabolism of endogenous and overexpressed APH1a and PEN2: evidence for artifactual involvement of the proteasome in the degradation of overexpressed proteins
Authors: Dunys Julie , Kawarai Toshitaka , Wilk Sherwin , St. George-Hyslop Peter , Alves Da Costa Cristine , Checler Frédéric
Published: March 2006

PS (presenilin)-dependent γ-secretase occurs as a high-molecular-mass complex composed of either PS1 or PS2 associated with Nct (nicastrin), PEN2 (presenilin enhancer 2 homologue) and APH1 (anterior pharynx defective 1 homologue). Numerous reports have documented the very complicated physical and functional cross-talk between these proteins that ultimately governs the biological activity of the γ-secretase, but very few studies examined the fate of the components of the complex. We show that, in both HEK-293 cells and the TSM1 neuronal cell line, the immunoreactivities of overexpressed myc-tagged-APH1a and -PEN2 were enhanced by the proteasome inhibitors ZIE and lactacystin, whereas a broad range of protease inhibitors had no effect. By contrast, proteasome inhibitors were totally unable to affect the cellular expression of endogenous APH1aL and PEN2 in HEK-293 cells, TSM1 and primary cultured cortical neurons. To explain this apparent discrepancy, we examined the degradation of myc-tagged-APH1a and -PEN2, in vitro, by cell extracts containing endogenous proteasome and by purified 20S proteasome. Strikingly, myc-tagged-APH1a and -PEN2 resist proteolysis by endogenous proteasome and purified 20S proteasome. We also show that endogenous PEN2 expression was drastically higher in wild-type than in PS- and Nct-deficient fibroblasts and was enhanced by proteasome inhibitors only in the two deficient cell systems. However, here again, purified 20S proteasome appeared unable to cleave endogenous PEN2 present in PS-deficient fibroblasts. The levels of endogenous APH1aL-like immunoreactivity were not modified by proteasome inhibitors and were unaffected by PS deficiency. Altogether, our results indicate that endogenous PEN2 and APH1aL do not undergo proteasomal degradation under physiological conditions in HEK-293 cells, TSM1 cells and fibroblasts and that the clearance of PEN2 in PS- and Nct-deficient fibroblasts is not mediated by 20S proteasome. Whether the 26S proteasome participates to PEN2 proteolysis in deficient fibroblasts remains to be established.
Quantitative mapping of oxidation-sensitive cysteine residues in SERCA in vivo and in vitro by HPLC–electrospray-tandem MS: selective protein oxidation during biological aging
Authors: Sharov Victor S. , Dremina Elena S. , Galeva Nadezhda A. , Williams Todd D. , Schöneich Christian
Published: March 2006

The selective reversible S-glutathiolation of specific SERCA (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase) cysteine residues represents a novel physiologic pathway of NO (nitric oxide)-dependent arterial smooth muscle relaxation [Adachi, Weisbrod, Pimentel, Ying, Sharov, Schöneich and Cohen (2004) Nat. Med. 10, 1200–1207]. This mechanism may be impaired through the irreversible oxidation of functionally important cysteine residues as a consequence of oxidative stress and aging. To establish whether in vivo aging and in vitro oxidation by peroxynitrite result in the loss of such functionally important cysteine residues of SERCA, we have developed and optimized a quantitative method to monitor the oxidation state of the individual SERCA cysteine residues using a maleimide-based fluorescence dye, TG1 (ThioGlo® 1), as a label for cysteine residues that have not been altered by oxidation and are not involved in disulphide bridges. A high efficiency for TG1 labelling of such residues and the chemical structure of cysteine–TG1 adducts were validated by MS analysis of model peptides, model proteins and rat skeletal muscle SERCA1. Tryptic peptides containing 18 out of a total of 24 cysteine residues were identified by HPLC–ESI (electrospray ionization)–MS/MS (tandem MS). Two cysteine residues, at positions 344 and 349, were detected in the form of an internal disulphide bridge, and another 16 were found to be labelled with TG1. Using HPLC–ESI–MS, we quantitatively mapped peroxynitrite oxidation of eight cysteine residues (positions 364, 417, 420, 498, 525, 674, 675 and 938), some of which are involved in the control of SERCA activity. Biological aging resulted in the partial modification of cysteine residues 377, 498, 525, 561, 614, 636, 674, 675, 774 and 938. Neither peroxynitrite exposure nor biological aging affected the apparent SERCA1 ATP affinity. Our data show an age-dependent loss of cysteine residues (approx. 2.8 mol of cysteine/mol of SERCA1), which may be partially responsible for the age-dependent decrease in the specific Ca2+-ATPase activity (by 40%).
Crystal structures of the editing domain of Escherichia coli leucyl-tRNA synthetase and its complexes with Met and Ile reveal a lock-and-key mechanism for amino acid discrimination
Authors: Liu Yunqing , Liao Jing , Zhu Bin , Wang En-Duo , Ding Jianping
Published: March 2006

aaRSs (aminoacyl-tRNA synthetases) are responsible for the covalent linking of amino acids to their cognate tRNAs via the aminoacylation reaction and play a vital role in maintaining the fidelity of protein synthesis. LeuRS (leucyl-tRNA synthetase) can link not only the cognate leucine but also the nearly cognate residues Ile and Met to tRNALeu. The editing domain of LeuRS deacylates the mischarged Ile–tRNALeu and Met–tRNALeu. We report here the crystal structures of ecLeuRS-ED (the editing domain of Escherichia coli LeuRS) in both the apo form and in complexes with Met and Ile at 2.0 Å, 2.4 Å, and 3.2 Å resolution respectively. The editing active site consists of a number of conserved amino acids, which are involved in the precise recognition and binding of the noncognate amino acids. The substrate-binding pocket has a rigid structure which has an optimal stereochemical fit for Ile and Met, but has steric hindrance for leucine. Based on our structural results and previously available biochemical data, we propose that ecLeuRS-ED uses a lock-and-key mechanism to recognize and discriminate between the amino acids. Structural comparison also reveals that all subclass Ia aaRSs share a conserved structure core consisting of the editing domain and conserved residues at the editing active site, suggesting that these enzymes may use a common mechanism for the editing function.
Induction of the permeability transition and cytochrome c release by 15-deoxy-Δ12,14-prostaglandin J2 in mitochondria
Authors: Landar Aimee , Shiva Sruti , Levonen Anna-Liisa , Oh Joo-Yeun , Zaragoza Corinne , Johnson Michelle S. , Darley-Usmar Victor M.
Published: March 2006

The electrophilic lipid 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is known to allow adaptation to oxidative stress in cells at low concentrations and apoptosis at high levels. The mechanisms leading to adaptation involve the covalent modification of regul-atory proteins, such as Keap1, and augmentation of antioxidant defences in the cell. The targets leading to apoptosis are less well defined, but mitochondria have been indirectly implicated in the mechanisms of cell death mediated by electrophilic lipids. To determine the potential of electrophilic cyclopentenones to induce pro-apoptotic effects in the mitochondrion, we used isolated liver mitochondria and demonstrated that 15d-PGJ2 promotes Ca2+-induced mitochondrial swelling and cytochrome c release. The mechanisms involved are consistent with direct modification of protein thiols in the mitochondrion, rather than secondary formation of reactive oxygen species or lipid peroxidation. Using proteomic analysis in combination with biotinylated 15d-PGJ2, we were able to identify 17 potential targets of the electrophile-responsive proteome in isolated liver mitochondria. Taken together, these results suggest that electrophilic lipid oxidation products can target a sub-proteome in mitochondria, and this in turn results in the transduction of the electrophilic stimulus to the cell through cytochrome c release.
Gene silencing of HIV chemokine receptors using ribozymes and single-stranded antisense RNA
Authors: Qureshi Amer , Zheng Richard , Parlett Terry , Shi Xiaoju , Balaraman Priyadhashini , Cheloufi Sihem , Murphy Brendan , Guntermann Christine , Eagles Peter
Published: March 2006

The chemokine receptors CXCR4 and CCR5 are required for HIV-1 to enter cells, and the progression of HIV-1 infection to AIDS involves a switch in the co-receptor usage of the virus from CCR5 to CXCR4. These receptors therefore make attractive candidates for therapeutic intervention, and we have investigated the silencing of their genes by using ribozymes and single-stranded antisense RNAs. In the present study, we demonstrate using ribozymes that a depletion of CXCR4 and CCR5 mRNAs can be achieved simultaneously in human PBMCs (peripheral blood mononuclear cells), cells commonly used by the virus for infection and replication. Ribozyme activity leads to an inhibition of the cell-surface expression of both CCR5 and CXCR4, resulting in a significant inhibition of HIV-1 replication when PBMCs are challenged with the virus. In addition, we show that small single-stranded antisense RNAs can also be used to silence CCR5 and CXCR4 genes when delivered to PBMCs. This silencing is caused by selective degradation of receptor mRNAs.
Characterization of the reversible phosphorylation and activation of ERK8
Authors: Klevernic Iva V. , Stafford Margaret J. , Morrice Nicholas , Peggie Mark , Morton Simon , Cohen Philip
Published: March 2006

ERK8 (extracellular-signal-regulated protein kinase 8) expressed in Escherichia coli or insect cells was catalytically active and phosphorylated at both residues of the Thr-Glu-Tyr motif. Dephosphorylation of the threonine residue by PP2A (protein serine/threonine phosphatase 2A) decreased ERK8 activity by over 95% in vitro, whereas complete dephosphorylation of the tyrosine residue by PTP1B (protein tyrosine phosphatase 1B) decreased activity by only 15–20%. Wild-type ERK8 expressed in HEK-293 cells was over 100-fold less active than the enzyme expressed in bacteria or insect cells, but activity could be increased by exposure to hydrogen peroxide, by incubation with the protein serine/threonine phosphatase inhibitor okadaic acid, or more weakly by osmotic shock. In unstimulated cells, ERK8 was monophosphorylated at Tyr-177, and exposure to hydrogen peroxide induced the appearance of ERK8 that was dually phosphorylated at both Thr-175 and Tyr-177. IGF-1 (insulin-like growth factor 1), EGF (epidermal growth factor), PMA or anisomycin had little effect on activity. In HEK-293 cells, phosphorylation of the Thr-Glu-Tyr motif of ERK8 was prevented by Ro 318220, a potent inhibitor of ERK8 in vitro. The catalytically inactive mutants ERK8[D154A] and ERK8[K42A] were not phosphorylated in HEK-293 cells or E. coli, whether or not the cells had been incubated with protein phosphatase inhibitors or exposed to hydrogen peroxide. Our results suggest that the activity of ERK8 in transfected HEK-293 cells depends on the relative rates of ERK8 autophosphorylation and dephosphorylation by one or more members of the PPP family of protein serine/threonine phosphatases. The major residue in myelin basic protein phosphorylated by ERK8 (Ser-126) was distinct from that phosphorylated by ERK2 (Thr-97), demonstrating that, although ERK8 is a proline-directed protein kinase, its specificity is distinct from ERK1/ERK2.
Post-translational and transcriptional regulation of DMT1 during P19 embryonic carcinoma cell differentiation by retinoic acid
Authors: Paradkar Prasad N. , Roth Jerome A.
Published: March 2006

Studies were performed to determine the regulation of DMT1 (divalent metal transporter 1) during RA (retinoic acid)-induced differentiation of P19 embryonic carcinoma cells. Protein and mRNA expression for the +/−IRE (iron response element) forms of DMT1, but not the 1A isoform, were down-regulated within the first few hours upon removal of RA, at which time the cells began to differentiate. The turnover of the +/−IRE isoforms of DMT1 protein during this period was found to be dependent on both the proteasomal and lysosomal pathways. Changes in mRNA levels were shown to be regulated by nitric oxide produced by the induction of neuronal nitric oxide synthase after removal of RA. Nitric oxide functions by inhibiting NF-κB (nuclear factor κB) nuclear translocation and the subsequent binding to the putative NF-κB response element (at −19 to −23) within the 1B promoter. Gel-shift analysis and chromatin immunoprecipitation assay indicated that nuclear NF-κB is capable of binding to this response element and that binding decreases during early stages of differentiation. Luciferase reporter gene assay demonstrated that a mutation in this binding domain leads to decreased activity. These results demonstrate that during neuronal differentiation of P19 cells, there is a decrease in specific isoforms of DMT1 via both post-translational and transcriptional mechanisms.