Exposure to the chlorofluorocarbon substitute 2,2-dichloro-1,1,1- trifluoroethane and the anesthetic agent halothane is associated with transient protein adduct formation in the heart.

Hydrochlorofluorocarbons (HCFCs) that are structural analogues of the anesthetic agent halothane may follow a common pathway of bioactivation and formation of adducts to cellular targets of distinct tissues. Exposure of rats to a single dose of HCFC 123 (2,2-dichloro- 1,1,1-trifluoroethane) or its structural analogue halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) in vivo resulted in the formation of one prominent trifluoroacetylated protein adduct (TFA-protein adduct) in the heart. In contrast, a variety of distinct TFA-protein adducts were formed in the liver and the kidney of the same animals. The TFA-protein adduct in the heart was processed rapidly; t1/2 of the intact TFA-protein adduct was less than 12 h.     

Proteomic-based identification of the APC-binding protein EB1 as a candidate of novel tissue biomarker and therapeutic target for colorectal cancer

Novel candidates of biomarker and therapeutic target in colorectal cancer (CRC) were investigated using a proteomic approach. The proteome of normal colorectal epithelial tissues was compared with that of the tumor ones in 59 CRC patients using two-dimensional difference gel electrophoresis. Of 3458 protein spots, 110 exhibited statistically significant (p<0.01) differences in intensity (more than 2.5-folds) between the normal and tumor tissue groups. Of 67 unique gene products that were identified for 105 of the 110 protein spots, we focused on the higher expression of the adenoma polyposis coli-binding protein EB1 (EB1). EB1 was originally discovered as a binding protein of APC, which is a tumor suppressor gene product, and the expression of EB1 has been associated with poor prognosis in several malignancies but not in CRC. Immunohistochemical analysis of the 132 CRC cases revealed that EB1 was overexpressed in tumor cells in correlation with poor prognosis. Suppression of EB1 by RNAi inhibited CRC cell proliferation and invasion. In this study, the overexpression of EB1 in CRC tissues correlating with prognosis, and its functional contribution to the malignant phenotypes of CRC cells are described. The present findings indicate that EB1 is a potential biomarker and therapeutic target in CRC.     

Identification of ribosomal protein S3a as a candidate for a novel PI 3-kinase target in the nucleus

Phosphatidylinositol 3,4,5-trisphosphate (PIP₃) is an important lipid second messenger that mediates various cell responses. We have searched for the nuclear PIP₃ binding proteins using PIP₃ analogue beads. A 33 kD protein was detected in this method, which was identified as ribosomal protein S3a by the mass spectrometric analysis. The recombinant S3a protein bound specifically to PIP₃. S3a localized not only in the cytosol but also in the nucleus. Interestingly, not cytosolic but nuclear S3a bound to PIP₃, suggesting different roles of S3a in the cytosol and the nucleus. This revised version was published online in August 2006 with corrections to the Cover Date.     

A novel RalGEF-like protein, RGL3, as a candidate effector for rit and Ras

The small GTPase Rit is a close relative of Ras, and constitutively active Rit can induce oncogenic transformation. Although the effector loops of Rit and Ras are highly related, Rit fails to interact with the majority of the known Ras candidate effector proteins, suggesting that novel cellular targets may be responsible for Rit transforming activity. To gain insight into the cellular function of Rit, we searched for Rit-binding proteins by yeast two-hybrid screening. We identified the C-terminal Rit/Ras interaction domain of a protein we have designated RGL3 (Ral GEF-like 3) that shares 35% sequence identity with the known Ral guanine nucleotide exchange factors (RalGEFs). RGL3, through a C-terminal 99-amino acid domain, interacted in a GTP- and effector loop-dependent manner with Rit and Ras. Importantly, RGL3 exhibited guanine nucleotide exchange activity toward the small GTPase Ral that was stimulated in vivo by the expression of either activated Rit or Ras. These data suggest that RGL3 functions as an exchange factor for Ral and may serve as a downstream effector for both Rit and Ras.     

The fate of norleucine as a replacement for methionine in protein synthesis.

In vivo synthesised protein with norleucine occupying one half of the normal methionine loci was prepared using a methionine auxotroph of Escherichia coli K12. The extent of charging of the analogue onto both tRNA_met species and subsequent incorporation into soluble protein was monitored with a double-labelling system comprising [G-³H]norleucine and [³⁵S]methionine. Further experiments established that norleucine can be formylated in vivo once charged onto the initiator tRNA^f_met. An N-terminal analysis of the crude soluble protein revealed that formylnorleucyl-tRNA^f_met can initiate protein synthesis and that the formyl group is then removed from the nascent polypeptide. We were also led to conclude that the N-terminal methionine-amino peptidase does not recognise the analogue in this position. Slow growth rates on the methionine analogue have been partly attributed to limiting levels of charged tRNA^m_met, resulting in turn from the inefficiency of norleucine charging by methionyl-tRNA synthetase. Finally no evidence has been found for the production of aberrant protein as a result of norleucine incorporation, implying that limited growth on the analogue is due to its inability to replace methionine as the precursor of S-adenosyl methionine.     

Dipeptidyl peptidase IV is a target for covalent adduct formation with the acyl glucuronide metabolite of the anti-inflammatory drug zomepirac

The nonsteroidal anti-inflammatory drug zomepirac (ZP) is metabolised to a chemically reactive acyl glucuronide conjugate (ZAG) which can form covalent adducts with proteins. In vivo, such adducts could initiate immune or toxic responses. In rats given ZP, the major band detected in liver homogenates by immunoblotting with a polyclonal ZP antiserum was at 110 kDa. This adduct was identified as ZP-modified dipeptidyl peptidase IV (DPP IV) by immunoblotting using the polyclonal ZP antiserum and monoclonal DPP IV antibodies OX-61 and 236.3. In vitro, ZAG, but not ZP itself, covalently modified recombinant human and rat DPP IV. Both monoclonal antibodies recognized DPP IV in livers from ZP- and vehicle-dosed rats. Confirmation that the 110 kDa bands which were immunoreactive with the ZP and DPP IV antibodies represented the same molecule was obtained from a rat liver extract reciprocally immunodepleted of antigens reactive with these two antibodies. Furthermore, immunoprecipitations with OX-61 antibody followed by immunolotting with ZP antiserum, and the reciprocal experiment, showed that both these antibodies recognised the same 110 kDa molecule in extracts of ZP-dosed rat liver. The results verify that DPP IV is one of the protein targets for covalent modification during hepatic transport and biliary excretion of ZAG in rats.     

Photoaptameric heterodimeric constructs as a new approach to enhance the efficiency of formation of photocrosslinks with a target protein

Using DNA aptamers selectively recognizing anion-binding exosites 1 and 2 of thrombin as a model, it has been demonstrated that their conjugation by a poly-(dT)-linker (ranging from 5 to 65 nucleotides (nt) in length) to produce aptamer heterodimeric constructs results into affinity enhancement. At the linker lengths ranged from 35 to 55 nt the apparent dissociation constants (K _D^app) measured using the optical biosensor Biacore-3000 for complexes of thrombin with the heterodimeric constructs reached minimum values (K _D^app) = 0.2–0.4 nM), which were approximately 30-fold less than for the complexes with the initial aptamers. A photoaptamer heterodimeric construct was designed connecting photoaptamer and aptamer sequences with the poly-(dT)-linker of 35 nt long. The photoaptamer used could form photo-induced cross-links with the exosite 2 of thrombin and the aptamer could bind to the exosite 1. The (K _D^app value for the photoaptamer construct was approximately 40-fold less than that for the primary photoaptamer (5.3 and 190 nM, respectively). Upon exposure of the equimolar mixtures of thrombin with the photoaptamer construct to the UV radiation at 308 nm the equal yield of the crosslinked complexes was observed at concentrations, which were lower by two orders of magnitude than in the case of the primary photoaptamer. It was found that concurrently with crosslinking to thrombin a photo-induced inactivation of the photoaptamer occurs presumably due to formation of the intermolecular crosslinking.     

The modified base J is the target for a novel DNA-binding protein in kinetoplastid protozoans

DNA from Kinetoplastida contains the unusual modified base beta-D-glucosyl(hydroxymethyl)uracil, called J. Base J is found predominantly in repetitive DNA and correlates with epigenetic silencing of telomeric variant surface glycoprotein genes in Trypanosoma brucei. We have now identified a protein in nuclear extracts of bloodstream stage T.brucei that binds specifically to J-containing duplex DNA. J-specific DNA binding was also observed with extracts from the kinetoplastids Crithidia fasciculata and Leishmania tarentolae. We purified the 90 kDa C.fasciculata J-binding protein 50 000-fold and cloned the corresponding gene from C.fasciculata, T.brucei and L.tarentolae. Recombinant proteins expressed in Escherichia coli demonstrated J-specific DNA binding. The J-binding proteins show 43-63% identity and are unlike any known protein. The discovery of a J-binding protein suggests that J, like methylated cytosine in higher eukaryotes, functions via a protein intermediate.     

Methylation levels of a novel genetic element, EgNB3 as a candidate biomarker associated with the embryogenic competency of oil palm

The association between DNA methylation status and embryogenic competency in oil palm tissue culture was examined through Representational Difference Analysis (RDA) approach, using methylation-sensitive restriction endonucleases. “Difference Products” (DPs) of RDA derived from palms of similar genetic backgrounds but exhibiting different embryogenesis rates during the regeneration process were isolated. The DPs were sequenced using a pyrosequencing platform. To our knowledge, this is the first study profiling partial HpaII methylation sites in oil palm young leaf tissues which are potentially associated with embryogenic amenability through a genomic subtractive approach. Quantitative real-time PCR analysis demonstrated that the methylation status of a novel fragment, EgNB3, was higher in highly embryogenic leaf explants compared to low embryogenesis rate materials. These differences are likely to be contributed by the 5′-^mCCGG-3′ and/or 5′-^mC^mCGG-3′ methylation patterns. Our data suggest that the differentially methylated site in EgNB3 has potential as a molecular biomarker for the screening of oil palm leaf explants for their embryogenic potentials.     

Expression of a novel FGF in the Xenopus embryo. A new candidate inducing factor for mesoderm formation and anteroposterior specification.

We have cloned and sequenced a new member of the fibroblast growth factor family from Xenopus laevis embryo cDNA. It is most closely related to both mammalian kFGF (FGF-4) and FGF-6 but as it is not clear whether it is a true homologue of either of these genes we provisionally refer to it as XeFGF (Xenopus embryonic FGF). Two sequences were obtained, differing by 11% in derived amino acid sequence, which probably represent pseudotetraploid variants. Both the sequence and the behaviour of in vitro translated protein indicates that, unlike bFGF (FGF-2), XeFGF is a secreted molecule. Recombinant XeFGF protein has mesoderm-inducing activity with a specific activity similar to bFGF. XeFGF mRNA is expressed maternally and zygotically with a peak during the gastrula stage. Both probe protection and in situ hybridization showed that the zygotic expression is concentrated in the posterior of the body axis and later in the tailbud. Later domains of expression were found near the midbrain/hindbrain boundary and at low levels in the myotomes. Because of its biological properties and expression pattern, XeFGF is a good candidate for an inducing factor with possible roles both in mesoderm induction at the blastula stage and in the formation of the anteroposterior axis at the gastrula stage.     

Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho.

The small GTP binding protein Rho is implicated in cytoskeletal responses to extracellular signals such as lysophosphatidic acid to form stress fibers and focal contacts. Here we have purified a Rho-interacting protein with a molecular mass of approximately 164 kDa (p164) from bovine brain. This protein bound to GTPgammaS (a non-hydrolyzable GTP analog).RhoA but not to GDP.RhoA or GTPgammaS.RhoA with a mutation in the effector domain (RhoAA37).p164 had a kinase activity which was specifically stimulated by GTPgammaS.RhoA. We obtained the cDNA encoding p164 on the basis of its partial amino acid sequences and named it Rho-associated kinase (Rho-kinase). Rho-kinase has a catalytic domain in the N-terminal portion, a coiled coil domain in the middle portion and a zinc finger-like motif in the C-terminal portion. The catalytic domain shares 72% sequence homology with that of myotonic dystrophy kinase and the coiled coil domain contains a Rho-interacting interface. When COS7 cells were cotransfected with Rho-kinase and activated RhoA, some Rho-kinase was recruited to membranes. Thus it is likely that Rho-kinase is a putative target serine/threonine kinase for Rho and serves as a mediator of the Rho-dependent signaling pathway.     

The distal nephron in the chick embryo as a target tissue for 1-alpha-25-dihydroxycholecalciferol

A histological and ultrastructural study as well as an autoradiographic analysis after injection of tritiated 1,25-dihydroxycholecalciferol (1,25-DHCC) were conducted on the distal convoluted tubules of chick embryos. Distal tubules in the embryo were shown to have the same spatial distribution as described for the adult kidney; they presented a convoluted portion located in the vicinity of the central intralobular veins and straight portions irradiating from this region towards the periphery. The epithelium in these tubules was well differentiated; its cells had numerous interdigitating folds in their lateral boundaries which were especially numerous at the basal ends. This device greatly increased the membrane surface available for interchange and was interpreted as an expression of active water and/or mineral transport. Nuclear concentration of radioactivity was found 2 h after injection of tritiated 1,25-DHCC in both the pars convoluta and the pars recta of the distal tubules. This concentration could be blocked by the previous administration of large amounts of nonradioactive 1,25-DHCC. These facts were interpreted as indicating that distal convoluted tubules in the chick embryo are functionally differentiated and contain target cells for 1,25-DHCC.     

Elongation factor 2 as a target for selective inhibition of protein synthesis in vitro by the novel aromatic biasamidine

By the formation of cGMP the NO-sensitive guanylyl cyclase plays a key role within the NO/cGMP signaling cascade involved in vascular regulation and neurotransmission. The prosthetic heme group of the enzyme acts as the NO sensor, and binding of NO induces conformational changes leading to an up to 200-fold activation of the enzyme. The unexpected fast dissociation half-life of NO of a few seconds is fast enough to account for the deactivation of the enzyme in biological systems. YC-1 and its analogues acting as NO sensitizers uncovered a new pharmacologically and conceivably physiologically relevant regulatory principle of the enzyme.Two existing isoforms of the heterodimeric guanylyl cyclase (₁₁, ₂₁) are known that are functionally indistinguishable. Up to now, the NO-sensitive guanylyl cyclase has been considered as a soluble enzyme. However, recent evidence about the ₂₁ isoform interacting with a PDZ domain of the postsynaptic scaffold protein PSD-95 suggests that the ₂ subunit directs a membrane association of this isoform. The interaction with PSD-95 locates the ₂₁ isoform in close proximity to the NO-generating NO synthase thereby enabling the NO sensor to respond to locally raised NO concentrations.     

Differential protein profile in the ear-punched tissue of regeneration and non-regeneration strains of mice: a novel approach to explore the candidate genes for soft-tissue regeneration

Wound repair/regeneration is a genetically controlled, complex process. In order to identify candidate genes regulating fast wound repair/regeneration in soft-tissue, the temporal protein profile of the soft-tissue healing process was analyzed in the ear-punched tissue of regeneration strain MRL/MpJ-Fas(lpr) (MRL) mice and non-regeneration strain C57BL/6J(B6) mice using surface-enhanced laser desorption and ionization (SELDI) ProteinChip technology. Five candidate proteins were identified in which responses of MRL to the ear punch were 2-4-fold different compared to that of B6. Their corresponding genes were predicted using an antigen-antibody assay validated mass-based approach. Most of the predicted genes are known to play a role or are likely to play a role in the wound repair/regeneration. Of the five candidate proteins, the amount of the 23560 Da protein in the ear-punched tissue was significantly correlated with the rate of ear healing in six representative strains of mice, making it a good candidate for fast wound repair/regeneration. We speculate that the increased concentration of the 23560 Da protein in the wound tissue could stimulate the expression of various growth-promoting proteins and consequently speed up the wound repair/regeneration processes. Here, we have shown that examination of protein expression profile using SELDI technology, coupled with database search, is an alternative approach to search for candidate genes for wound repair/regeneration. This novel approach can be implemented in a variety of biological applications.     

Sequence and expression of a wheat gene that encodes a novel protein associated with pathogen defense.

Wheat (Triticum aestivum) exhibits local acquired resistance to the powdery mildew pathogen Erysiphe graminis f. sp. tritici. The resistant state can be induced by a preinoculation with the nonhost pathogen E. g.f. sp. hordei, the barley powdery mildew, and is accompanied by the activation of putative defense genes. Here, we report the sequence of a pathogen-induced gene, WIR1a, and a corresponding cDNA, WIR1, that encode novel defense-related proteins of 88 and 85 amino acids, respectively. Analysis of the primary structure of these proteins predicts them to be integral membrane proteins with extracytoplasmic C-terminal domains rich in proline and glycine, through which the proteins possibly interact with the cell wall.     

Characterization of a novel GTPase-activating protein associated with focal adhesions and the actin cytoskeleton

In the present study we characterize a novel RhoGAP protein (RC-GAP72) that interacts with actin stress fibers, focal adhesions, and cell-cell adherens junctions via its 185-amino acid C-terminal region. Overexpression of RC-GAP72 in fibroblasts induces cell rounding with partial or complete disruption of actin stress fibers and formation of membrane ruffles, lamellipodia, and filopodia. RC-GAP72 mutant truncated downstream of the GTPase-activating protein (GAP) domain retains the ability to stimulate membrane protrusions but fails to affect stress fiber integrity or induce cell retraction. A mutant protein consisting of the C terminus of RC-GAP72 and lacking the GAP domain does not exert any visible effect on cellular morphology. Inactivation of the GAP domain by a point mutation does not abolish the effect of RC-GAP72 on actin stress fibers but moderates its capability to induce membrane protrusions. Our data imply that the cytoskeletal localization of RC-GAP72 and its interaction with GTPases are essential for its effect on the integrity of actin stress fibers, whereas the induction of lamellipodia and filopodia depends on the activity of the GAP domain irrespective of binding to the actin cytoskeleton. We propose that RC-GAP72 affects cellular morphology by targeting activated Cdc42 and Rac1 GTPases to specific subcellular sites, triggering local morphological changes. The overall physiological functions of RC-GAP72 are presently unknown, yet our data suggest that RC-GAP72 plays a role in regulating cell morphology and cytoskeletal organization.     

Identification and characterization of a novel extracellular matrix protein nephronectin that is associated with integrin alpha8beta1 in the embryonic kidney

The epithelial-mesenchymal interactions required for kidney organogenesis are disrupted in mice lacking the integrin alpha8beta1. None of this integrin's known ligands, however, appears to account for this phenotype. To identify a more relevant ligand, a soluble integrin alpha8beta1 heterodimer fused to alkaline phosphatase (AP) has been used to probe blots and cDNA libraries. In newborn mouse kidney extracts, alpha8beta1-AP detects a novel ligand of 70-90 kD. This protein, named nephronectin, is an extracellular matrix protein with five EGF-like repeats, a mucin region containing a RGD sequence, and a COOH-terminal MAM domain. Integrin alpha8beta1 and several additional RGD-binding integrins bind nephronectin. Nephronectin mRNA is expressed in the ureteric bud epithelium, whereas alpha8beta1 is expressed in the metanephric mesenchyme. Nephronectin is localized in the extracellular matrix in the same distribution as the ligand detected by alpha8beta1-AP and forms a complex with alpha8beta1 in vivo. Thus, these results strongly suggest that nephronectin is a relevant ligand mediating alpha8beta1 function in the kidney. Nephronectin is expressed at numerous sites outside the kidney, so it may also have wider roles in development. The approaches used here should be generally useful for characterizing the interactions of novel extracellular matrix proteins identified through genomic sequencing projects.     

Phosphorylation of basic fibroblast growth factor by a protein kinase associated with the outer surface of a target cell.

A protein kinase capable of phosphorylating basic fibroblast growth factor (FGF) can be localized on the outer cell surface of human hepatoma cells (SK-Hep cells). The addition of [gamma-32P]ATP, but not H3(32)PO4, results in a rapid (less than 10 min) incorporation of 32P into exogenously added basic FGF. The reaction is time and concentration dependent (apparent Km, 170 nM) and is stimulated by the addition of cAMP (EC50, 0.5 microM), but not the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate. There is also no tyrosine protein kinase detected on the cell surface. The inhibition of basic FGF binding to its low and/or high affinity sites decreases the phosphorylation of basic FGF by the ecto-protein kinase. Accordingly, pretreatment of cells with heparinase for 30 min or coincubation with heparin (0.1-10 micrograms/ml) decreases phosphorylation in a dose-dependent manner. Furthermore, the addition of a nonphosphorylatable peptide analog of basic FGF ([Val112] basic FGF-(106-146)NH2) that can compete with basic FGF binding to cells prevents the phosphorylation of basic FGF. Together, these observations suggest that 1) exogenous basic FGF must associate with its low and/or high affinity binding sites to be phosphorylated, and 2) the kinase is cAMP dependent and associated with the outer cell surface, and support the hypothesis that phosphorylation may regulate the activity and/or bioavailability of the growth factor.