Smurf2 is a TRAF2 binding protein that triggers TNF-R2 ubiquitination and TNF-R2-induced JNK activation

TRAF2 plays a central role in TNF-induced signalling to NF-κB and JNK/p38 MAPK. To better understand the molecular mechanisms that mediate this dual function of TRAF2, we performed a yeast two-hybrid screening for TRAF2 interacting proteins using the Sos recruitment system. This resulted in the identification of the E3 ubiquitin ligase Smurf2 as a TRAF2 binding protein. TRAF2 overexpression was shown to trigger Smurf2 ubiquitination and the formation of a TNF-R2/Smurf2 complex. Smurf2 on its turn promoted TNF-R2 ubiquitination and the relocalization of TNF-R2 as well as TRAF2 to a detergent-insoluble cell fraction. This was associated with enhanced TNF-R2-induced JNK activation, whereas TNF-R2-induced NF-κB activation remained unaffected. These results suggest an important role for Smurf2 binding to TRAF2 in determining specific signalling outputs of TNF-R2.     

Degradation of 2-chlorobenzoate by Pseudomonas cepacia 2CBS

A bacterium was isolated from water by enrichment on 2-chlorobenzoate as sole source of carbon and energy. Based on morphological and physiological properties, this microorganism was assigned to the species Pseudomonas cepacia. The organism was designated Pseudomonas cepacia 2CBS. During growth on 2-chlorobenzoate, the chlorine substituent was released quantitatively, and a small amount of 2,3-dihydroxybenzoate accumulated in the culture medium. Mutants of Pseudomonas cepacia 2CBS were induced by treatment with N-methyl-N'-nitro-N-nitrosoguanidine. Some of these mutants produced catechol from 2-chlorobenzoate. Other mutants accumulated the meta-cleavage product of catechol, 2-hydroxy-cis,cis-muconic acid semialdehyde. In crude cell-free extracts of Pseudomonas cepacia 2CBS, an enzyme was detected which catalysed the conversion of 2-chlorobenzoate to catechol. Molecular oxygen, NADH and exogenous Fe2+ were required for activity. Stoichiometric amounts of chloride were released. Experiments with 18O2 revealed that both oxygen atoms in the hydroxyl groups of the product were derived from molecular oxygen. Thus, the enzyme catalysing the conversion of 2-chlorobenzoate was identified as 2-chlorobenzoate 1,2-dioxygenase (1,2-hydroxylating, dehalogenating, decarboxylating). 2-Chlorobenzoate 1,2-dioxygenase from Pseudomonas cepacia 2CBS was shown to be a multicomponent enzyme system. The activities of catechol 2,3-dioxygenase and catechol 1,2-dioxygenase were detected in crude cell-free extracts. The activity of catechol 2,3-dioxygenase was 60 times higher than the activity of catechol 1,2-dioxygenase, indicating that catechol is mainly degraded via meta-cleavage in Pseudomonas cepacia 2CBS. No enzyme was found which converted 2,3-dihydroxybenzoate, suggesting that this compound is a dead-end metabolite of 2-chlorobenzoate catabolism. A pathway for the degradation of 2-chlorobenzoate by Pseudomonas cepacia 2CBS is proposed.     

The inhibitory activities of 2-acetamido-2,3-dideoxy-D-hex-2-enonolactones on 2-acetamido-2-deoxy-beta-D-glucosidase.

Treatment of 2-acetamido-2-deoxy-D-mannono-1,4-lactone with dicyclohexylamine in ethanolic solution afforded an unsaturated 1,4-lactone, 2-acetamido-2,3-dideoxy-D-erythro-hex-2-enono-1,4-lactone (1), in good yield. 2-Acetamido-2,3-dideoxy-D-threo-hex-2-enono-1,4-lactone (2) was similarly prepared from 2-acetamido-2-deoxy-D-galactono-1,4-lactone. An unsaturated 1,5-lactone, 2-acetamido-2,3-dideoxy-D-threo-hex-2-enono-1,5-lactone (4), was obtained through the oxidation of 2-acetamido-2-doexy-4,6-0-isopropylidene-D-galactopyranose with silver carbonate on Celite, followed by mild hydrolysis. The inhibitory activity of four isomeric 2-acetamido-2,3-dideoxy-D-hex-2-enonolactones [1, 2, 4, and 2-acetamido-2,3-dideoxy-D-erythro-hex-2-enono-1,5-lactone (3)] was assayed against 2-acetamido-2-deoxy-beta-D-glucosidase from bull epididymis. Only the erythro lactones 1 and 3 are weak competitive inhibitors, whereas the threo lactones 2 and 4 are practically inactive. The 1,4-lactone 1 inhibited 2-acetamido-2-deoxy-beta-D-glucosidase more strongly than the 1,5-lactone 3. The lactones 1-4 were found to be quite stable in aqueous solution or under inhibitory-assay conditions. In addition, two 2-acetamido-2-deoxy-D-glycals, 2-acetamido-1,5-anhydrohex-1-enitol (7) were tested; both are 10 times as active as 1.     

Indole-2-carboxamidines as novel NR2B selective NMDA receptor antagonists

A novel series of indole-2-carboxamidine derivatives was prepared and identified as NR2B selective NMDA receptor antagonists. The influence of the substituents on the indole skeleton as well as the substitution of the benzyl moiety on the biological activity of the compounds was studied. Compound 5a was po active in the formalin test in mouse.     

Contra-IL 2; a suppressor lymphokine that inhibits IL 2 activity

Suppressive activity of culture supernatant of AS-9 (AS-9 CS), a T cell hybridoma line that was derived from fusion of BW5147 thymoma and splenic T cells of anti-lymphocyte serum-treated C3H mice, was analyzed. AS-9 CS inhibited allogeneic cytotoxic T lymphocyte (CTL) generation as well as T cell proliferation to alloantigens and mitogens, but failed to inhibit B cell response to lipopolysaccharide or growth of tumor and fibroblast cells. Although addition of AS-9 CS to the allogeneic sensitization culture as late as on day 2 of incubation resulted in maximal inhibiton of CTL generation, removal of AS-9 CS on day 3 of incubation abolished its inhibitory effect. Addition of purified IL 2 together with AS-9 CS to the allogeneic sensitization cultures only partially abrogated the suppression. Experiments with IL 2-dependent cytotoxic T cell line (CTLL) showed that AS-9 CS suppressed the IL 2-induced proliferation of CTLL. Preincubation of AS-9 CS with CTLL removed its inhibitory effect on CTL generation. These results indicate that AS-9 CS interferes with the mechanism of T cell activation by IL 2. On this basis, AS-9 CS was named contra-IL 2.     

2-fluoro-ATP: a toxic metabolite of 9-beta-D-arabinosyl-2-fluoroadenine

Murine P388 cells incubated in vitro with the anticancer drug arabinosyl 2-fluoroadenine accumulate its 5'-triphosphate, F-araATP, as the major phosphorylated metabolite. A new chromatographically separate metabolite that accumulated to levels 10% of that of F-araATP was identified as 2-fluoro-ATP, by the following criteria. 1. The metabolite coeluted with the authentic compound on anion-exchange HPLC. 2. Dephosphorylation of the metabolite yielded a compound that was chromatographically identical to 2-fluoroadenosine. 3. The compound was sensitive to NaIO4 oxidation. Cellular incubation experiments indicated that 2-fluoroadenine, but not arabinosyl 2-fluorohypoxanthine, was the likely intermediate in the formation of 2-fluoro-ATP.     

Identification of an H2O2 permeable PIP aquaporin in barley and a serine residue promoting H2O2 transport

A barley (Hordeum vulgare) plasma membrane type aquaporin, HvPIP2;5, was identified as an H₂O₂ permeable aquaporin among 21 barley and rice PIPs examined in the heterologous expression system using Saccharomyces cerevisiae. Four TIPs were also detected as H₂O₂‐transporting aquaporins among 15 barley and rice TIPs. Influx of H₂O₂ into yeast cells expressing HvPIP2;5 was determined with a florescent‐dye‐based assay. Indirect immunofluorescence indicated that the expression of HvPIP2;5 protein was ubiquitous in root tissues, and was also weakly observed in leaf epidermal cells and cells in the vascular bundle. Point mutated variants of HvPIP2;5 were generated by the site‐directed mutagenesis. Growth assays of yeast cells expressing these mutated HvPIP2;5 proteins suggested that Ser‐126 in HvPIP2;5 has a large impact on H₂O₂ transport with a minor influence on the HvPIP2;5‐mediated water transport.     

FHL2通过相互作用抑制Id2的功能活性

分化抑制蛋白2（Id2）通过抑制碱性螺旋-环-螺旋（bHLH）类转录因子的功能活性调控多种组织细胞的分化发育,并参与人类多种肿瘤的发生与进展.Id2相互作用蛋白可能调控其翻译后的功能活性．本研究以HLH结构域缺失的Id2作为诱饵蛋白,采用酵母双杂交方法对MCF-7 cDNA文库进行筛选,识别了1个新的Id2相互作用蛋白FHL2 (属于LIM蛋白家族的一员),哺乳动物双杂交实验系统验证了Id2与FHL2之间的相互作用,同时证实,该作用不依赖于Id2中的HLH结构域;GST-pulldown、免疫共沉淀方法,进一步证实FHL2/Id2之间的相互作用;免疫荧光共定位实验结果证实,FHL2/Id2相互作用主要发生在细胞核内;共转染实验结果发现,FHL2通过相互作用阻抑了Id2对bHLH类转录因子E47的功能抑制活性．总之,本研究识别了1个新的Id2相互作用蛋白FHL2,通过直接的相互作用,FHL2抑制了Id2的功能活性,FHL2可能参与调控Id2介导的细胞分化与发育过程,并可能参与肿瘤的发生与进展．     

Degradation of 2-sec-butylphenol: 3-sec-butylcatechol,2-hydroxy-6-oxo-7-methylnona-2,4-dienoic acid,and 2-methylbutyric acid as intermediates

Pseudomonas sp. strain HBP1 Prp, a mutant of strain HBP1 that was originally isolated on 2-hydroxybiphenyl, was able to grow on 2-sec-butylphenol as the sole carbon and energy source. During growth on 2-sec-butylphenol, 2-methylbutyric acid transiently accumulated in the culture medium. Its concentration reached a maximum after 20 hours and was below detection limit at the end of the growth experiment. The first three enzymes of the degradation pathway — a NADH-dependent monooxygenase, a metapyrocatechase, and ameta-fission product hydrolase — were partially purified. The product of the the monooxygenase reaction was identified as 3-sec-butylcatechol by mass spectrometry. This compound was a substrate for the metapyrocatechase and was converted to 2-hydroxy-6-oxo-7-methylnona-2,4-dienoic acid which was identified by gas chromatography-mass spectrometry of its trimethylsilyl-derivative. The cofactor independentmeta-cleavage product hydrolase used 2-hydroxy-6-oxo-7-methylnona-2,4-dienoic acid as a substrate. All three enzymes showed highest activities for 2-hydroxybiphenyl and its metabolites, respectively, indicating that 2-sec-butylphenol is metabolized via the same pathway as 2-hydroxybiphenyl.     

Quantification of calsequestrin 2 (CSQ2) in sheep cardiac muscle and Ca2+-binding protein changes in CSQ2 knockout mice

Calsequestrin 2 (CSQ2) is generally regarded as the primary Ca2+-buffering molecule present inside the sarcoplasmic reticulum (SR) in cardiac cells, but findings from CSQ2 knockout experiments raise major questions about its role and necessity. This study determined the absolute amount of CSQ2 present in cardiac ventricular muscle to gauge its likely influence on SR free Ca2+ concentration ([Ca2+]) and maximal Ca2+ capacity. Ventricular tissue from hearts of freshly killed sheep was examined by SDS-PAGE without any fractionation, and CSQ2 was detected by Western blotting; this method avoided the >90% loss of CSQ2 occurring with usual fractionation procedures. Band intensities were compared against those for purified CSQ2 run on the same blots. Fidelity of quantification was verified by demonstrating that CSQ2 added to homogenates was detected with equal efficacy as purified CSQ2 alone. Ventricular tissue from sheep (n=8) contained 24±2 μmol CSQ2/kg wet wt. Total Ca2+ content of the ventricular tissue, measured by atomic absorption spectroscopy, was 430±20 μmol/kg (with SR Ca2+ likely<250 μmol/kg) and displayed a linear correlation with CSQ2 content, with gradient of ～10 Ca2+ per CSQ2. The large amount of CSQ2 bestows the SR with a high theoretical maximal Ca2+-binding capacity (～1 mmol Ca2+/kg ventricular tissue, assuming a maximum of ～40 Ca2+ per CSQ2) and would keep free [Ca2+] within the SR relatively low, energetically favoring Ca2+ uptake and reducing SR leak. In mice with CSQ2 ablated, histidine-rich Ca2+-binding protein was upregulated ～35% in ventricular tissue, possibly in compensation.     

Regulation of Sprouty2 stability by mammalian Seven-in-Absentia homolog 2

Mammalian Sprouty (Spry) gene expression is rapidly induced upon activation of the FGF receptor signaling pathway in multiple cell types including cells of mesenchymal and epithelial origin. Spry2 inhibits FGF-dependent ERK activation and thus Spry acts as a feedback inhibitor of FGF-mediated proliferation. In addition, Spry2 interacts with the ring-finger-containing E3 ubiquitin ligase, c-Cbl, in a manner that is dependent upon phosphorylation of Tyr55 of Spry2. This interaction results in the poly-ubiquitination and subsequent degradation of Spry2 by the proteasome. Here, we describe the identification of another E3 ubiquitin ligase, human Seven-in-Absentia homolog-2 (SIAH2), as a Spry2 interacting protein. We show by yeast two-hybrid analysis that the N-terminal domain of Spry2 and the ring finger domain of SIAH2 mediated this interaction. Co-expression of SIAH2 resulted in proteasomal degradation of Spry1, 2, and to a lesser extent Spry4. The related E3 ubiquitin-ligase, SIAH1, had little effect on Spry2 protein stability when co-expressed. Unlike c-Cbl-mediated degradation of Spry2, SIAH2-mediated degradation was independent of phosphorylation of Spry2 on Tyr55. Spry2 was also phosphorylated on Tyr227, and phosphorylation of this residue was also dispensable for SIAH2-mediated degradation of Spry2. Finally, co-expression of SIAH2 with Spry2 resulted in a rescue of FGF2-mediated ERK phosphorylation. These data suggest a novel mechanism whereby Spry2 stability is regulated in a manner that is independent of tyrosine phosphorylation, and provides an addition level of control of Spry2 protein levels.     

Interleukin-2-collagen chimeric protein which liberates interleukin-2 upon collagenolysis

Interleukin-2 (IL-2) is a potent activator of cellular immunity and has been utilized as an immunotherapeutic agent. We stably immobilized human IL-2 to collagen by covalently binding it to the N-terminus of human type III collagen (3A1) as IL2-3A1 chimeric protein using recombinant technology. The present study was aimed at liberating IL-2 from the immobilized chimeric protein by treating the chimera with bacterial collagenase. These IL2-3A1 chimeras were synthesized in insect cells which had been infected with baculovirus vectors carrying IL2-3A1 cDNA. The IL2-3A1 protein produced was shown to be in a pepsin-resistant triple helical structure and exhibited IL-2 activity to a similar extent as IL-2 itself. IL2-3A1 could be immobilized on the surface of plastic dishes by incubating it in the dishes. The IL-2 region of the immobilized IL2-3A1 was liberated to culture media by collagenase treatment and this freed IL-2 stimulated the growth of lined T cells. Thus, IL2-3A1 chimeric protein could be utilized as an IL-2 deliverer whose T cell mitogenic activity can be liberated by a collagenolytic environment.     

Dual oxidase-2 has an intrinsic Ca2+-dependent H2O2-generating activity

Duox2 (and probably Duox1) is a glycoflavoprotein involved in thyroid hormone biosynthesis, as the thyroid H2O2 generator functionally associated with Tpo (thyroperoxidase). So far, because of the impairment of maturation and of the targeting process, transfecting DUOX into nonthyroid cell lines has not led to the expression of a functional H2O2-generating system at the plasma membrane. For the first time, we investigated the H2O2-generating activity in the particulate fractions from DUOX2- and DUOX1-transfected HEK293 and Chinese hamster ovary cells. The particulate fractions of these cells stably or transiently transfected with human or porcine DUOX cDNA demonstrate a functional NADPH/Ca2+-dependent H2O2-generating activity. The immature Duox proteins had less activity than pig thyrocyte particulate fractions, and their activity depended on their primary structures. Human Duox2 seemed to be more active than human Duox1 but only half as active as its porcine counterpart. TPO co-transfection produced a slight increase in the enzymatic activity, whereas p22(phox), the 22-kDa subunit of the leukocyte NADPH oxidase, had no effect. In previous studies on the mechanism of H2O2 formation, it was shown that mature thyroid NADPH oxidase does not release O2*- but H2O2. Using a spin-trapping technique combined with electron paramagnetic resonance spectroscopy, we confirmed this result but also demonstrated that the partially glycosylated form of Duox2, located in the endoplasmic reticulum, generates superoxide in a calcium-dependent manner. These results suggest that post-translational modifications during the maturation process of Duox2 could be implicated in the mechanism of H2O2 formation by favoring intramolecular superoxide dismutation.     

Acid Hydrolysis of Methyl 2-Amino-2-deoxy-d-glucopyranosides and Methyl d-Glucuronides

In connection with the behavior on hydrolysis of mucopolysaccharides, acid hydrolysis of methyl d-glucopyranosides, methyl 2-amino-2-deoxy-d-glucopyranosides (hydrochlorides as well as N-substituted derivatives), and methyl d-glucuronides was carried out. The difference in hydrolysis rate of methyl 2-amino-2-deoxy-d-glucopyranosides was ascribable to that of the substituents on the amino group, whereas hydrolysis rate of methyl d-glucuronides was dependent on their ring structures. The possible behaviors in acid hydrolysis of glycosidic linkages in mucopolysaccharides are discussed.     

Synthesis of [2S-2-H]- and [2R-2-H]hexadecanoic acids

[2S-2-²H]- and [2R-2-²H]hexadecanoic acids were synthesized in overall yields of 59–67%. Methyl(2R)-2-hydroxyhexadecanoate, from the acid produced by Hansenula sydowiorum, was converted to the p-toluenesulphonate, reduced to trideutero alcohol with lithium aluminium deuteride and oxidized to [2S-2-²H]hexadecanoic acid. Methyl (2S)-2-chlorohexadecanoate, which was a by-product of tosylation and was also prepared by chlorinatioon of the hydroxy ester with thionyl chloride, on reduction and oxidation as before gave [2R-2-²H]-hexadecanoic acid. Intermediates were fully characterized, isotopic purity was 97% and optical purity was maintained throughout the syntheses. Attempts to reduce the tosyl or chloro groups, only, with sodium borodeuteride gave low yields probably due to preferential reduction of the ester group; 1,2-epoxyhexadecane was obtained from the tosylate and 2-chlorohexadecan-1-ol from the chloro ester.     

5'-Esters of 2'-deoxyadenosine and 2-chloro-2'-deoxyadenosine with cell differentiation-provoking agents

Phenylacetic and retinoic acids are carboxyacidic cell differentiating agents displaying anticancer activities. We report on a new class of compounds including the 5'-esters of 2'-deoxyadenosine (dA) or 2-chloro-2'-deoxyadenosine (cladribine, 2CdA) and the aforementioned acids. The rationale behind the synthesis of these esters was that if they are hydrolyzed inside the lymphoid cells, either dA will be removed from the intracellular environment by deamination, or 2CdA will be phosphorylated and accumulated. In either case targetted delivery of the differentiating agent to the lymphoid cells may be envisaged. The said compounds were synthesized by the Mitsunobu procedure employing triphenylphosphine and azadicarboxylic acid esters, and their stability was tested against various esterases. Esters of dA and 2CdA with phenylacetic acids were found to be resistant to enzymatic hydrolysis, whereas those with retinoic acids were efficiently hydrolyzed by commercially available hepatic esterase as well as by esterases present in the blood plasma and in diluted human lymphocyte lysate. Susceptibility to enzymatic hydrolysis was found to be a prerequisite of cytotoxic and/or differentiating activity of these esters in leukemic cell lines.