Oxidation of Bovine β-Casein by Hypochlorite

We recently observed two 2,4-dinitrophenylhydrazine (DNPH)-reactive proteins of 40 and 120 kDa in the bronchoalveolar lavage fluids of rats exposed to >95% O₂ for 48 h. The N-terminal sequences of these proteins were both identical over 16 amino acids with rat β-casein, which, in addition to its more common association with milk, is produced by cytotoxic T-lymphocytes, and has been found to have proinflammatory properties. Because of the inflammatory response that accompanies hyperoxic lung injury, we investigated the oxidation of bovine β-casein by HOCl. Following exposure to HOCl at 4°C for 15 min, derivatization with DNPH, washing, and digestion with trypsin, the resultant peptides were separated by reverse-phase HPLC. One peptide isolated from a peak absorbing at 365 nm was identified as AVP(Y*)PQR, corresponding to amino acids 177–183 of bovine β-casein. Analysis of the peptide by both electrospray and matrix assisted laser desorption ionization (MALDI) mass spectrometry identified a molecular ion MH⁺ of 1008.5 Da, which represents an increase of 178 Da from the calculated monoisotopic MH⁺ of the unmodified peptide of 830.45 Da. Daughter ion spectra of the doubly charged parent ion of the peptide further support the oxidation of the tyrosine to the quinone methide, with subsequent conversion to the corresponding hydrazone with DNPH. A second pair of products were identified as arising from oxidation of Y¹⁹³ within the tryptic peptide constituted by amino acids 184–202, and the corresponding chymotryptic cleavage side product, 191–202. Exposure of β-casein to increasing amounts of HOCl revealed that M and Y residues were the most susceptible, although bovine β-casein contains no C, and a single W, which would not be detected by our methods. The approach described in the present report can be used to evaluate the contributions of distinct mechanisms of oxidation in other experimental or pathological models. © 1997 Elsevier Science Inc.     

Ligand-induced internalization of TNF receptor 2 mediated by a di-leucin motif is dispensable for activation of the NFκB pathway

Endocytosis is an important mechanism to regulate tumor necrosis factor (TNF) signaling. In contrast to TNF receptor 1 (TNFR1; CD120a), the relevance of receptor internalization for signaling as well as the fate and route of internalized TNF receptor 2 (TNFR2; CD120b) is poorly understood. To analyze the dynamics of TNFR2 signaling and turnover at the plasma membrane we established a human TNFR2 expressing mouse embryonic fibroblast cell line in a TNFR1^−/−/TNFR2^−/− background. TNF stimulation resulted in a decrease of constitutive TNFR2 ectodomain shedding. We hypothesized that reduced ectodomain release is a result of TNF/TNFR2 complex internalization. Indeed, we could demonstrate that TNFR2 was internalized together with its ligand and cytoplasmic binding partners. Upon endocytosis the TNFR2 signaling complex colocalized with late endosome/lysosome marker Rab7 and entered the lysosomal degradation pathway. Furthermore, we identified a di-leucin motif in the cytoplasmic part of TNFR2 suggesting clathrin-dependent internalization of TNFR2. Internalization defective TNFR2 mutants are capable to signal, i.e. activate NFκB, demonstrating that the di-leucin motif dependent internalization is dispensable for this response. We therefore propose that receptor internalization primarily serves as a negative feed-back to limit TNF responses via TNFR2.     

Urothelial transdifferentiation to prostate epithelia is mediated by paracrine TGF-β signaling

The embryonic urogenital sinus mesenchyme (UGM) induces prostate epithelial morphogenesis in development. The molecular signals that drive UGM-mediated prostatic induction have not been defined. We hypothesized that the TGF-β signaling directed the prostatic induction. UGM from TGF-β type II receptor stromal conditional knockout mice (Tgfbr2^fspKO) or control mice (Tgfbr2^{floxE2/floxE2}) was recombined with wild-type adult mice bladder urothelial cells. The resulting urothelium associated with Tgfbr2^{floxE2/floxE2} UGM was instructively differentiated into prostatic epithelium, as expected. In contrast, the urothelium associated with Tgfbr2^fspKO UGM permissively maintained the phenotype of bladder epithelial cells. Microarray analysis of UGM tissues suggested the down-regulation of multiple Wnt ligands and the up-regulation of the Wnt antagonist, Wif 1, by the Tgfbr2^fspKO UGM compared with Tgfbr2^{floxE2/floxE2} UGM. The overexpression of Wif-1 by wild-type UGM resulted in the inhibition of prostatic induction. These data suggest that the stromal TGF-β activity mediated by paracrine Wnt is necessary for the induction of prostatic differentiation. As Wnt ligands mediate differentiation and maintain the stem cell phenotype, the contribution of mouse stem cells and somatic cells to prostatic epithelium in the tissue recombination models was tested. The directed differentiation of mouse embryonic stem cells by UGM is suggested by a threshold number of mouse stem cells required in prostatic differentiation. To determine the contribution of somatic cells, the adult bladder epithelial compartment was labeled with green-fluorescent vital dye (CMFDA) and the stem-like cells marked by bromodeoxyuridine (BrdU) label-retention. The resulting prostatic epithelia of the tissue recombinants maintained the CMFDA dye, suggesting minimal cell division. Thus, the UGM can induce endoderm-derived epithelia and stem cells to form prostate through a transdifferentiation mechanism that requires stromal TGF-β signaling to mediate epithelial Wnt activity.     

Spatial regulation of cell adhesion in the Drosophila wing is mediated by Delilah, a potent activator of βPS integrin expression

In spite of our conceptual view of how differential gene expression is used to define different cell identities, we still do not understand how different cell identities are translated into actual cell properties. The example discussed here is that of the fly wing, which is composed of two main cell types: vein and intervein cells. These two cell types differ in many features, including their adhesive properties. One of the major differences is that intervein cells express integrins, which are required for the attachment of the two wing layers to each other, whereas vein cells are devoid of integrin expression. The major signaling pathways that divide the wing to vein and intervein domains have been characterized. However, the genetic programs that execute these two alternative differentiation programs are still very roughly drawn. Here we identify the bHLH protein Delilah (Dei) as a mediator between signaling pathways that specify intervein cell-fate and one of the most significant realizators of this fate, βPS integrin. Dei's expression is restricted to intervein territories where it acts as a potent activator of βPS integrin expression. In the absence of normal Dei activity the level of βPS integrin is reduced, leading to a failure of adhesion between the dorsal and ventral wing layers and a consequent formation of wing blisters. The effect of Dei on βPS expression is not restricted to the wing, suggesting that Dei functions as a general genetic switch, which is turned on wherever a sticky cell-identity is determined and integrin-based adhesion is required.     

Survivin is a guardian of the intestinal stem cell niche and its expression is regulated by TGF-β

As an inhibitor of apoptosis (IAP) family member, Survivin is known for its role during regulation of apoptosis. More recently its function as a cell cycle regulator has become evident. Survivin was shown to play a pivotal role during embryonic development and is highly expressed in regenerative tissue as well as in many cancer types. We examined the function of Survivin during mouse intestinal organogenesis and in gut pathophysiology. We found high expression of Survivin in experimentally induced colon cancer in mice but also in colon tumors of humans. Moreover, Survivin was regulated by TGF-β and was found to be highly expressed during mucosal healing following intestinal inflammation. We identified that expression of Survivin is essential early on in life, as specific deletion of Survivin in Villin expressing cells led to embryonic death around day 12 post coitum. Together with our recent study on the role of Survivin in the gut of adult mice our data demonstrate that Survivin is an essential guardian of embryonic gut development and adult gut homeostasis protecting the epithelium from cell death promoting the proliferation of intestinal stem and progenitor cells.     

Multiple elements of the S 2 ?-RNase promoter from potato ( Solanum tuberosum L.) are required for cell type-specific expression in transgenic potato and tobacco

A functional analysis of the promoter of the S ₂ -RNase gene from potato was performed in transgenic potato and tobacco plants, using a deletion series of S ₂ -RNase promoter GUS fusions. A detailed histochemical and quantitative analysis of the transgenic tobacco plants revealed that S ₂ promoter fragments ranging in size from 5.6 kb in length down to 0.2 kb mediate a weak developmentally regulated expression in the pistil, and strong ectopic expression in pollen. In the pistil, different expression patterns were seen depending on the transformant, the predominant one being characterised by expression in the stigma and the transmitting tract of the style, whereas a few plants showed expression exclusively either in the stigma or in the stylar transmitting tissue. All transformants also showed GUS expression in the placental epidermis of the ovary. Two sequences that are conserved between the potato S ₁ -RNase and S ₂ -RNase promoters, termed motif I and motif III, are located in a fragment of the S ₂ promoter extending from position −200 to bp −100, and motif II, located between bp −498 and −480, was identified on the basis of sequence comparisons between pistil-specific promoters. Motif II was found to be dispensible for pistil-specific and for pollen-specific expression. Two submotifs, A and B, were identified within motif I. Both were essential for expression in the pistil but only B was necessary for expression in pollen. Although motif III has a similar bipartite structure and sequence to motif I, it was not sufficient to confer either pollen- or pistil-specific expression. However, deletion of motif III abolished pollen-specific expression in transient expression experiments, suggesting that an interaction between the two sequence motifs may be needed to specify cell type-specific expression. In transgenic potato the S ₂ -RNase promoter also mediates expression in pollen and in the pistil; however, significantly fewer plants showed expression than in tobacco, with most plants also exhibiting GUS expression in other tissues. Received: 7 August 1997 / Accepted: 8 September 1997     

Synthesis and transporter binding properties of (R)-2β,3β- and (R)-2α,3α-diaryltropanes

(R)-2-Aryl-2-tropinone (9) was synthesized from (R)-2-carbomethoxy-3-tropinone (5) and was used as the key intermediate for the synthesis of (R)-2β,3β- and (R)-2α,3α-diaryltropanes. Inhibition of radioligand binding studies at the dopamine, serotonin, and norepinephrine transporters showed that the (R)-3β-(4-methylphenyl)-2β-phenyltropane (3b, RTI-422) possessed an IC₅₀ value of 1.96 nM at the dopamine transporter and was highly selective for this transporter relative to the serotonin and norepinephrine transporters.     

Suppression of RhoG activity is mediated by a syndecan 4–synectin–RhoGDI1 complex and is reversed by PKCα in a Rac1 activation pathway

Fibroblast growth factor 2 (FGF2) is a major regulator of developmental, pathological, and therapeutic angiogenesis. Its activity is partially mediated by binding to syndecan 4 (S4), a proteoglycan receptor. Angiogenesis requires polarized activation of the small guanosine triphosphatase Rac1, which involves localized dissociation from RhoGDI1 and association with the plasma membrane. Previous work has shown that genetic deletion of S4 or its adapter, synectin, leads to depolarized Rac activation, decreased endothelial migration, and other physiological defects. In this study, we show that Rac1 activation downstream of S4 is mediated by the RhoG activation pathway. RhoG is maintained in an inactive state by RhoGDI1, which is found in a ternary complex with synectin and S4. Binding of S4 to synectin increases the latter''s binding to RhoGDI1, which in turn enhances RhoGDI1''s affinity for RhoG. S4 clustering activates PKCα, which phosphorylates RhoGDI1 at Ser⁹⁶. This phosphorylation triggers release of RhoG, leading to polarized activation of Rac1. Thus, FGF2-induced Rac1 activation depends on the suppression of RhoG by a previously uncharacterized ternary S4–synectin–RhoGDI1 protein complex and activation via PKCα.     

Nuclear translocation of glutathione S-transferase π is mediated by a non-classical localization signal

Glutathione S-transferase π (GSTπ), a member of the GST family of multifunctional enzymes, is highly expressed in human placenta and involved in the protection of cellular components against electrophilic compounds or oxidative stress. We have recently found that GSTπ is expressed in the cytoplasm, mitochondria, and nucleus in some cancer cells, and that the nuclear expression of GSTπ appears to correlate with resistance to anti-cancer drugs. Although the mitochondrial targeting signal of GSTπ was previously identified in the amino-terminal region, the mechanism of nuclear translocation remains completely unknown. In this study, we find that the region of GSTπ195–208 is critical for nuclear translocation, which is mediated by a novel and non-classical nuclear localization signal. In addition, using an in vitro transport assay, we demonstrate that the nuclear translocation of GSTπ depends on the cytosolic extract and ATP. Although further experiments are needed to understand in depth the precise mechanism of nuclear translocation of GSTπ, our results may help to establish more efficient anti-cancer therapy, especially with respect to resistance to anti-cancer drugs.     

Restricted tissue-specific but correct developmental expression mediated by a short human α1AT promoter fragment in transgenic mice

The tissue-specific and developmental pattern of expression controlled by the proximal promoter (position –348 to+15) derived from the human -1-antitrypsin (h1AT) gene was studied in transgenic mice. The short promoter segment was linked to the chloramphenicol acetyltransferase (CAT) reporter gene. The transgene showed highly specific expression in the liver and the correct developmental pattern of regulation. Interestingly, this short promoter targets expression to the liver with a greater specificity than that reported for larger 1AT promoter fragments.     

Early planarian brain regeneration is independent of blastema polarity mediated by the Wnt/β-catenin pathway

Analysis of anteroposterior (AP) axis specification in regenerating planarian flatworms has shown that Wnt/β-catenin signaling is required for posterior specification and that the FGF-like receptor molecule nou-darake (ndk) may be involved in restricting brain regeneration to anterior regions. The relationship between re-establishment of AP identity and correct morphogenesis of the brain is, however, still poorly understood. Here we report the characterization of two axin paralogs in the planarian Schmidtea mediterranea. Although Axins are well known negative regulators of Wnt/β-catenin signaling, no role in AP specification has previously been reported for axin genes in planarians. We show that silencing of Smed-axin genes by RNA interference (RNAi) results in two-tailed planarians, a phenotype previously reported after silencing of Smed-APC-1, another β-catenin inhibitor. More strikingly, we show for the first time that while early brain formation at anterior wounds remains unaffected, subsequent development of the brain is blocked in the two-tailed planarians generated after silencing of Smed-axin genes and Smed-APC-1. These findings suggest that the mechanisms underlying early brain formation can be uncoupled from the specification of AP identity by the Wnt/β-catenin pathway. Finally, the posterior expansion of the brain observed following Smed-ndk RNAi is enhanced by silencing Smed-APC-1, revealing an indirect relationship between the FGFR/Ndk and Wnt/β-catenin signaling systems in establishing the posterior limits of brain differentiation.     

Highly efficient gene expression of a fibrinolytic enzyme (subtilisin DFE) in Bacillus subtilis mediated by the promoter of α-amylase gene from Bacillus amyloliquefaciens

Subtilisin DFE is a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4. The promoter and signal peptide-coding sequence of alpha-amylase gene from B. amyloliquefaciens was cloned and fused to the sequence coding for pro-peptide and mature peptide of subtilisin DFE. This hybrid gene was inserted into the Escherichia coli/Bacillus subtilis shuttle plasmid vector, pSUGV4. Recombinant subtilisin DFE gene was successfully expressed in B. subtilis WB600 with a fibrinolytic activity of 200 urokinase units ml(-1).     

Characterization of an acid-labile, thermostable β-glycosidase from Thermoplasma acidophilum

A recombinant putative β-galactosidase from Thermoplasma acidophilum was purified as a single 57 kDa band of 82 U mg⁻¹. The molecular mass of the native enzyme was 114 kDa as a dimer. Maximum activity was observed at pH 6.0 and 90°C. The enzyme was unstable below pH 6.0: at pH 6 its half-life at 75°C was 28 days but at pH 4.5 was only 13 h. Catalytic efficiencies decreased as p-nitrophenyl(pNP)-β-d-fucopyranoside (1067) > pNP-β-d-glucopyranoside (381) > pNP-β-d-galactopyranoside (18) > pNP-β-d-mannopyranoside (11 s⁻¹ mM⁻¹), indicating that the enzyme was a β-glycosidase.     

Inhibition of adenylate cyclase activity in the goldfish melanophore is mediated by α 2-adrenoceptors and a pertussis toxin-sensitive GTP-binding protein

The signal-transduction system that mediates the melanosome-aggregating response in melanophores of the black-moor goldfish, Carassius auratus, was investigated by examining the inhibition of adenylate cyclase activity mediated by -adrenoceptors in cultured cells. When the melanophores were incubated with 1 mmol·l^-1 3-isobutyl-1-methylxanthine for 5 min, the intracellular level of cyclic adenosine-3,5-monophosphate increased two- to three-fold. Norepinephrine at 100 nmol·l^-1 and naphazoline at 1 mol·l^-1 inhibited the 3-isobutyl-1-methylxanthine-induced accumulation of cyclic adenosine-3,5-monophosphate in the cells in both the presence and the absence of isoproterenol, a -adrenergic agonist. Methoxamine and phenylephrine also reduced the extent of accumulation of cyclic adenosine-3,5-monophosphate, but only when they were present at relatively high concentrations (above 100 mol·l^-1). The range of concentrations at which norepinephrine inhibited the accumulation of cyclic adenosine-3,5-monophosphate was consistent with the range at which it induced the aggregation of melanosomes. Pretreatment of the cells with pertussis toxin (1 g·ml^-1) for 15 h or treatment with 100 nmol·l^-1 yohimbine (an ₂-adrenergic antagonist) inhibited the effects of the -adrenergic agonists on both the aggregation of melanosomes and the 3-isobutyl-1-methylxanthine-induced accumulation of cyclic adenosine-3,5-monophosphate, but prazosin (an ₁adrenergic antagonist) at 100 nmol·l^-1 was not inhibitory. These results indicate that the melanosome-aggregating response of the goldfish melanophore is induced mainly via inhibition of the activity of adenylate cyclase, which occurs as result of stimulation of a pathway that involves ₁adrenergic and a inhibitory GTP-binding protein.Abbreviations A-kinase cAMP-dependent protein kinase - BSS balanced salts solution - CaM calmodulin - cAMP cyclic adenosine-3,5-monophosphate - Clo clonidine - EDTA ethylenediaminetetra-acetic acid - G-protein GTP-binding protein - HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid - IBMX 3-isobutyl-1-methylxanthine - IP₃ inositol 1,4,5-trisphosphate Mex, methoxamine - MSH melanocyte-stimulating hormone - Nap naphazoline - NE norepinephrine - Oxy oxymetazoline - Phe phenylephrine - PTX pertussis toxin