Interaction between chicken protein tyrosine phosphatase 1 (CPTP1)-like rat protein phosphatase 1 (PTP1) and p60(v-src) in v-src-transformed Rat-1 fibroblasts

CPTP1 is a nontransmembrane chicken protein tyrosine phosphatase having 92% sequence homology to the corresponding 321 amino acids of human protein tyrosine phosphatase 1B (HPTP1B). Using anti-CPTP1 antibody, we identified CPTP1-like rat PTP1 of 51 kDa in Rat-1 and v-src-transformed Rat-1 fibroblasts. Here we show that CPTP1-like rat PTP1 binds to p60(v-src) in vivo and CPTP1 also can associate with p60(v-src) in cell lysate of v-src- transformed Rat-1 fibroblasts. Interaction between HPTP1B-type PTPs, CPTP1-like rat PTP1 and CPTP1, and p60(v-src) was reduced by vanadate treatment for 13 h due to down regulation of the protein level of p60(v-src) in vivo. Interestingly, CPTP1-like rat PTP1 was coimmunoprecipitated with a 70-kDa protein which has a possibility to be tyrosine- phosphorylated by p60(v-src) in v-src-transformed Rat-1 fibroblasts. These results suggest that HPTP1B-type PTPs may play an important role in p60(src) dependent signal pathway in eucaryotic cells.     

Nucleophilic substitution reaction of benzyl benzenesulfonates with anilines in MeOH-MeCN mixtures-I : Effects of substituent and solvent on the transition-state structure

Kinetic studies on nucleophilic substitution reaction of benzyl tosylates with anilines are reported. The reaction was found to proceed via a dissociative S_N2 mechanism with less than 50 % bond formation and extensive bond breaking at the transition state. It was found that positive charge development at the benzylic carbon is substantial and para-substituent effect on the substrate is predominantly of resonance type. Bond formation is shown to be favored by a better nucleophile, by an electron withdrawing group on the substrate and by the more polar(higher MeCN content) solvent. The substrate, nucleophile and solvent were found to follow the RSP.     

Significantly enhanced reactivities of the nucleophilic substitution reactions in ionic liquid

We have investigated the reactivities of various metal fluorides in the nucleophilic fluorination of 2-(3-methanesulfonyloxypropyl)naphthalene (1) as a model compound in the presence of 1-n-butyl-3-methylimidazolium tetrafluoroborate ([bmim][BF(4)]). The higher periodic alkali metal fluorides demonstrate greater reactivity. The fluorination using CsF among the alkali metal fluorides was completed in 20 min, affording the desired product 2-(3-fluoropropyl)naphthalene (2a, 95%) without any byproducts. However, the fluorinations using alkali earth, transition, and low periodic alkali metal fluorides under the same conditions occurred rarely or not at all. We have also carried out the various facile nucleophilic substitutions such as halogenations, acetoxylation, nitrilation, and alkoxylations of mesyloxyalkane 1 and 2-(3-bromopropyl)naphthalene (6) at the primary aliphatic position using the potassium halides, acetate, cyanide, and alkoxides, respectively, in the presence of ionic liquids. These reactions provided the desired products, such as 2-(3-halopropyl)naphthalenes 5-7 (95% for Cl, 96% for Br, and 93% for I), 2-(3-acetoxypropyl)naphthalene (8, 95%), 2-(3-cyanopropyl)naphthalene (9, 93%), and 2-(3-methoxypropyl)naphthalene (10, 92%).     

Synthesis of aliphatic ketones from allylic alcohols through consecutive isomerization and chelation-assisted hydroacylation by a rhodium catalyst

An allylic alcohol, utilized as a precursor for an aliphatic aldehyde, reacted with olefins to afford aliphatic ketones in the presence of RhCl(PPh(3))(3), 2-amino-4-picoline, aniline, and benzoic acid through a tandem reaction of an isomerization and a chelation-assisted hydroacylation.     

Pheromone synthesis. Part 244: Synthesis of the racemate and enantiomers of (11Z,19Z)-CH503 (3-acetoxy-11,19-octacosadien-1-ol), a new sex pheromone of male Drosophila melanogaster to show its (S)-isomer and racemate as bioactive

The enantiomers of (11Z,19Z)-3-acetoxy-11,19-octacosadien-1-ol were synthesized from the enantiomers of 3,4-epoxy-1-butanol PMB ether. Its racemate was also synthesized. Its (S)-isomer and racemate were shown to possess the same pheromone activity as CH503, a long-lived inhibitor of male courtship in Drosophila melanogaster, although the racemate was less active.     

Cytosolic accumulation of gammaH2AX is associated with tropomyosin-related kinase A-induced cell death in U2OS cells

Tropomyosin-related kinase A (TrkA) plays an important role in cell survival, differentiation, and apoptosis in various neuronal and nonneuronal cell types. Here we show that TrkA overexpression by the Tet-On system mimics NGF-mediated activation pathways in the absence of nerve growth factor (NGF) stimulation in U2OS cells. In addition, p53 upregulation upon DNA damage was inhibited by TrkA, and p21 was upregulated by TrkA in a p53-independent manner. TrkA overexpression caused cell death by interrupting cell cycle progression, and TrkA-induced cell death was diminished in the presence of its specific inhibitor GW441756. Interestingly, TrkA-mediated cell death was strongly related to gammaH2AX production and poly (ADP-ribose) polymerase cleavage in the absence of DNA damage inducer. In this study, we also reveal that gammaH2AX production by TrkA is blocked by TrkA kinase inhibitors K-252a and GW441756, and it is also significantly inhibited by JNK inhibitor SP600125. Moreover, reduction of cell viability by TrkA was strongly suppressed by SP600125 treatment, suggesting a critical role of JNK in TrkA-induced cell death. We also found that gammaH2AX and TrkA were colocalized in cytosol in the absence of DNA damage, and the nuclear localization of gammaH2AX induced by DNA damage was partly altered to cytosol by TrkA overexpression. Our results suggest that the abnormal cytosolic accumulation of gammaH2AX is implicated in TrkA-induced cell death in the absence of DNA damage.     

3-Dimensional cell culture for on-chip differentiation of stem cells in embryoid body

This paper proposes a microfluidic device for the on-chip differentiation of an embryoid body (EB) formed in a microwell via 3-dimensional cultures of mouse embryonic carcinoma (EC) cells. The device adjusted the size of the EB by fluid volume, differentiated the EB by chemical treatment, and evaluated its effects in EC cells by on-chip immunostaining. A microfluidic resistance network was designed to control the size of the embryoid body. The duration time and flow rate into each microwell regulated the initial number of trapped cells in order to adjust the size of the EB. The docked cells were aggregated and formed a spherical EB on the non-adherent surface of the culture chip for 3 days. The EC cells in the EB were then differentiated into diverse cell lineages without attachment for an additional 4 days; meanwhile, retinoic acid (RA) was applied without serum to direct the cells into early neuronal lineage. On-chip immunostaining of the EB in the microwell with a neuronal marker was conducted to assess the differentiation-inducing ability of RA. The effect of RA on neuronal differentiation was analyzed with confocal microscopic images of the TuJ1 marker. The RA-treated cells expressed more neuronal markers and appeared as mature neuronal cells with long neurites. The fluorescence intensity of the TuJ1 in the RA-treated EB was twice that observed in the non-treated EB on day 5. It was demonstrated that the pre-screening of inducing chemicals on the early neuronal differentiation of EC cells in a single microfluidic chip was indeed feasible. This chip is expected to constitute a useful tool for assessing the early differentiation of ES cells without attachment, and is also expected to prove useful as an anti-cancer drug test platform for the cytotoxicity assay with cellular spheroids.