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Enucleation is the process whereby the nucleus is extruded from the erythroblast during late stage mammalian erythropoiesis. However, the specific signaling pathways involved in this process remain unclear. To better understand the mechanisms underlying erythroblast enucleation, we investigated erythroblast enucleation using both the spleens of adult mice with phenylhydrazine‐induced anemia and mouse fetal livers. Our results indicated that both iron‐bound transferrin (holo‐Tf) and the small‐molecule iron transporter hinokitiol with iron ions (hinokitiol plus iron) promote hemoglobin synthesis and the enucleation of mouse spleen‐derived erythroblasts. Although an antitransferrin receptor 1 (TfR1) monoclonal antibody inhibited both enucleation and hemoglobin synthesis promoted by holo‐Tf, it inhibited only enucleation, but not hemoglobin synthesis, promoted by hinokitiol plus iron. Furthermore, siRNA against mouse TfR1 were found to suppress the enucleation of mouse fetal liver‐derived erythroblasts, and the endocytosis inhibitor MitMAB inhibited enucleation, hemoglobin synthesis, and the internalization of TfR1 promoted by both types of stimuli. Collectively, our results suggest that TfR1, iron ions, and endocytosis play important roles in mouse erythroblast enucleation.

Aim Previously, we reported that mice deficient in most of the Zfp521 coding region (Zfp521Δ/Δ mice) displayed abnormal behaviors, including hyperlocomotion and lower anxiety. In this study, we aimed to elucidate the involvement and mechanisms of monoamine variation. Main methods First, we compared the levels of dopamine (DA), noradrenaline (NA), and serotonin in the brains of Zfp521Δ/Δ and Zfp521+/+ mice using enzyme-linked immunosorbent assay. Next, we elucidated the mechanisms using quantitative PCR and Western Blotting. Additionally, we administered inhibitory drug to the mice and performed behavioral tests. Key findings Our results showed that the DA level decreased and the NA level increased in Zfp521Δ/Δ mice. We found that ZFP521 suppresses the expression of dopamine β-hydroxylase (DBH), which converts DA into NA. We also demonstrated that paired homeodomain transcription factor 2 and early growth response protein-1, which are the transcription factors for Dbh, were involved in the upregulation of Dbh by ZFP521. The administration of nepicastat, a specific inhibitor of DBH, attenuated the abnormal behaviors of Zfp521Δ/Δ mice. Significance These results suggest that the lack of ZFP521 upregulates the expression of DBH, which leads to a decrease in the DA level and an increase in the NA level in the brain, resulting in abnormal behaviors.

AimsSTAT3 is a key modulator of activation and differentiation of macrophages. But it is still unknown if deficiency of STAT3 activates macrophages to destroy erythrocytes by phagocytosis. We generated STAT3 conditional knockout mice by crossing floxed STAT3 mice with Tie2 promoter-driven Cre-recombinase transgenic mice and clarified that Stat3 plays a critical role in the formation and activation of macrophages.Main methodsBlood cell count, reticulocyte count, serum lactate dehydrogenase, erythropoietin, iron and ferritin concentration, and life span of the erythrocytes in Tie2 promoter-driven STAT3 conditional knockout mice were analyzed. To explore the erythropoietic function of the mice, we subjected them to brief hemolytic anemia by injecting them intraperitoneally with phenylhydrazine. The fragility of erythrocytes was examined by scanning electron microscopy and osmotic tolerance test.Key findingsThe conditional knockout mice had mild normocytic anemia. They also displayed higher lactate dehydrogenase, ferritin and erythropoietin concentration, higher reticulocyte count, and a shorter lifespan of erythrocytes compared with wild-type controls. These data suggest that destruction of erythrocytes and secondary blood formation were accelerated in the STAT3 conditional knockout mice. It didn't appear due to the fragility of erythrocytes. A few of the conditional knockout mice suddenly developed acute severe anemia, high body temperature and massive splenomegaly, and died within 2weeks after the onset of anemia.SignificanceThis study provided evidence that STAT3 have a critical role in the destruction of erythrocytes by resident macrophages in the spleen.

The extract from Panax ginseng has been reported to improve the microcirculation in various organs. However, the mechanisms underlying this phenomenon are still poorly understood. In the present study, using the rheological properties of erythrocytes as an index, we have screened the components of Panax ginseng extract and identified Rg(2) and Rh(1) as the active ingredients. These two ginsenosides prevented the oxidative stress-induced elevation of erythrocyte suspension viscosity and the impairment of erythrocyte elongation in response to shear stress. Rg(2) and Rh(1) ginsenosides did not have antioxidant activity in an aqueous phase and did not inhibit the peroxidation of membrane lipids, either. However, they inhibited the oxidation-induced decrease of SH-groups in band 3 (anion exchanger-1), one of the important structural proteins of the erythrocyte membrane, but not in other structural proteins: bands 1 and 2 (spectrins), band 4.2 or band 5 (actin). These results suggest that ginsenosides Rg(2) and Rh(1) protect the rheological functions of erythrocytes against oxidative stress by preventing the oxidation of SH-groups in band 3 protein.

Nicastrin interacts with gamma-secretase complex components predominantly via the N-terminal third of the transmembrane domain. The authentic transmembrane domain is critically required for the interaction with gamma-secretase complex components and for formation of an active gamma-secretase complex. In this study, we have identified a novel alternatively spliced transcript of nicastrin in human brain tissue. This transcript (NCSTN-DeltaE16) lacks exon 16 of nicastrin mRNA, which leads to deletion of 71 amino acids just upstream of its transmembrane domain. Its expression pattern was analyzed in the hippocampus of patients with pathologically diagnosed Alzheimer disease (cases) and non-Alzheimer dementia (controls). In patients with the APOE-epsilon4 allele, the frequency of Alzheimer disease appeared to be increased in the NCSTN-DeltaE16-positive group, but the association was not statistically significant. In conclusion, the expression of NCSTN-DeltaE16 transcript may confer some additional risk for developing Alzheimer disease beyond the risk due to ApoE-epsilon4 allele. Further investigation in larger scale population would be necessary to address its potential implication in Alzheimer disease.

A 58-kDa protein was detected in Xenopus egg lysate by SDS-PAGE and immunoblotting with an antibody raised against adaptor protein Shc, a well known tyrosine kinase substrate in numerous biological events. Tyrosine phosphorylation of the Xenopus Shc protein (p58 xShc) was found to increase 2.3 +/- 0.4-fold (n = 3) upon fertilization. Pretreatment of eggs with the tyrosine kinase inhibitor genistein effectively blocked the fertilization-dependent phosphorylation. Tyrosine phosphorylation of p58 xShc was also observed when eggs were activated parthenogenetically by an integrin-interacting RGDS-peptide which is known to cause egg activation accompanied by intracellular calcium release. On the other hand, other egg-activating treatments such as electrical shock and calcium ionophore, which directly induce the elevation of intracellular calcium, did not show such an effect. It is also suggested that the phosphorylated p58 xShc may play a role unique to the egg activation process because we found that there was no increase of Shc-Grb2 complex after fertilization. These results demonstrate that p58 xShc is a substrate of egg tyrosine kinases which may be activated by sperm-egg interaction and suggest that the phosphorylated p58 xShc may act upstream of the calcium-dependent pathway of egg activation.

Zinc finger protein 521 (ZFP521) regulates a number of cellular processes in a wide range of tissues, such as osteoblast formation and adipose commitment and differentiation. In the field of neurobiology, it is reported to be an essential factor for transition of epiblast stem cells into neural progenitors in vitro. However, the role of ZFP521 in the brain in vivo still remains elusive. To elucidate the role of ZFP521 in the mouse brain, we generated mice lacking exon 4 of the ZFP521 gene. The birth ratio of our ZFP521 Δ/Δ mice was consistent with Mendel's laws. Although ZFP521 Δ/Δ pups had no apparent defect in the body and were indistinguishable from ZFP521+/+ and ZFP521 +/Δ littermates at the time of birth, ZFP521 Δ/Δ mice displayed significant weight reduction as they grew, and most of them died before 10 weeks of age. They displayed abnormal behavior, such as hyper-locomotion, lower anxiety and impaired learning, which correspond to the symptoms of schizophrenia. The border of the granular cell layer of the dentate gyrus in the hippocampus of the mice was indistinct and granular neurons were reduced in number. Furthermore, Sox1-positive neural progenitor cells in the dentate gyrus and cerebellum were significantly reduced in number. Taken together, these findings indicate that ZFP521 directly or indirectly affects the formation of the neuronal cell layers of the dentate gyrus in the hippocampus, and thus ZFP521 Δ/Δ mice displayed schizophrenia-relevant symptoms. ZFP521 Δ/Δ mice may be a useful research tool as an animal model of schizophrenia.

In the previous study (Sato K.-I. et al. (1997) FEBS Lett. 410, 136–140), we showed that the phosphorylation of Shc protein by c-Src is dependent on the binding of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) to the PTB domain of Shc. In this study, we demonstrate that, in contrast to c-Src, v-Src and epidermal growth factor (EGF) receptor can phosphorylate Shc in a PtdIns(4,5)P2-independent manner and at different phosphorylation sites. To determine the phosphorylation sites in Shc, we used mutant Shc proteins in which tyrosine residues (Y) 317 and/or 239 and 240 were replaced by phenylalanine residues (F). We found that Y317F Shc but not Y239/240F or Y239/240/317F Shc was phosphorylated by c-Src. The reaction was PtdIns(4,5)P2-dependent and inhibited by the addition of PTB domain of Shc. On the other hand, v-Src and EGF receptor were able to phosphorylate both Y317F and Y239/240F but not Y239/240/317F Shc in a PtdIns(4,5)P2-independent manner. These results highlight the difference between c-Src and v-Src or EGF receptor and suggest that c-Src can phosphorylate predominantly on Tyr239/240 of Shc only when Shc PTB domain is bound to PtdIns(4,5)P2.

We have examined the interaction between c-Src and epidermal growth factor (EGF) receptor in A431 cells. c-Src was found exclusively in the Triton X-100-solubilized particulate fraction and activated up to 3-fold within 1 min after EGF treatment of the cells. Association between c-Src and EGF receptor was detected by immunoprecipitation of c-Src followed by immunoblotting with anti-EGF receptor antibody. The c-Src-EGF receptor complex was found in both EGF-treated and untreated cells, but an augmented complex formation was observed in EGF-treated cells. We have isolated the complex by DEAE-cellulose column chromatography and found that a site-specific anti-c-Src antibody, which was raised against a synthetic peptide corresponding to residues 413 to 431 of human c-Src, did not recognize the c-Src protein in the complex, while other c-Src-specific antibodies tested did. Incubation of the complex with this synthetic peptide resulted in a partial dissociation of the complex. These results suggest that the specific region of c-Src is involved in the association with EGF receptor.

Protein kinase C (PKC) was partially purified from Xenopus laevis oocytes by ammonium sulfate fractionation followed by DEAE-cellulose and hydroxyapatite column chromatography. In the latter chromatography, two distinct PKC activities were identified. Both PKC fractions contained an 80 kDa protein which was recognized by three antisera raised against the conserved regions of mammalian PKC. However, specific antisera against alpha, beta I, beta II, and gamma-subspecies of rat PKC did not recognize the protein. Kinetic properties of the Xenopus PKCs were very similar to those of the rat alpha PKC, and only a subtle difference was found in the mode of activation by arachidonic acid. When oocytes were treated with the tumor promoter, phorbol 12-myristate 13-acetate, one of the Xenopus PKCs was found to disappear very rapidly, while the other remained unchanged up to 2 hr.

Erythrocytes are very susceptible to oxidative stress, having a high content of intracellular oxygen and hemoglobin. In the present study, exposure to oxidative stress resulted in a significant impairment of erythrocyte membrane functions, such as deformability and anion exchange. Band 3 protein, also known as anion exchanger-1, plays an important role in these two functions. We show that oxidative stress activated caspase-3 inside the erythrocytes, which resulted in band 3 protein cleavage. Interestingly, inhibition of the caspase-3 with its specific inhibitor not only suppressed the digestion of band 3 protein, but also blunted the functional damage to erythrocytes, such as deformability and anion exchange, without changing the level of peroxidation of membrane lipids. These results provide experimental evidence that activation of caspase-3 plays an important role in the oxidative stress-induced impairment of membrane functions of erythrocytes.

Apoptosis is defined by several unique morphological nuclear changes, such as chromatin condensation and nuclear fragmentation1. These changes are triggered by the activation of a family of cysteine proteases called caspases2,3, and caspase-activated DNase (CAD/DFF40)4,5 and lamin protease (caspase-6)6,7 have been implicated in some of these changes. CAD/DFF40 induces chromatin condensation in purified nuclei, but distinct caspase-activated factor(s) may be responsible for chromatin condensation8. Here we use an in vitro system to identify a new nuclear factor, designated Acinus, which induces apoptotic chromatin condensation after cleavage by caspase-3 without inducing DNA fragmentation. Immunodepletion experiments showed that Acinus is essential for apoptotic chromatin condensation in vitro, and an antisense study revealed that Acinus is also important in the induction of apoptotic chromatin condensation in cells.

To explore the structural basis required for the holoenzyme formation of cAMP-dependent protein kinase, we have prepared rabbit anti-peptide antibodies that can block the holoenzyme formation without affecting the catalytic activity of the enzyme. The antibodies were raised against a specific site in the catalytic (C)-subunit, termed IDA (Inter-DFG-APE) region, which lies between the kinase subdomains VII and VIII. Although the C-subunit immunoprecipitated with anti-IDA antibodies could not form a stable complex with regulatory (R)-subunit, it was still susceptible to inhibition by the R-subunit or by PKI, a specific inhibitor peptide containing a pseudosubstrate site. These results indicate that there exists an IDA regionmediated interaction between the R- and C-subunits, which is distinct from that mediated through the substrate site and substrate binding site. In accordance with this idea, association of synthetic IDA peptides with the R-subunit was directly demonstrated by resonance mirror analysis. The calculated association constants of IDA peptides were high enough to suggest a possible involvement of the IDA region in the initial step of holoenzyme formation.

In the previous study [Sato et al. (1995) Biochem. Biophys. Res. Commun. 210, 844-851], we found that c-Src was associated with epidermal growth factor (EGF) receptor and activated upon EGF treatment in A431 cells. In the present study, we investigated the phosphorylation of EGF receptor by c-Src in the c-Src-EGF receptor complex. We have focused our attention to tyrosine residue 845 (Y845) of EGF receptor as a candidate for the phosphorylation site. A synthetic peptide containing Y845, named Y845 peptide, which corresponds to residue 837 to 856 of EGF receptor, was found to be phosphorylated by c-Src and used to provide the standard phosphopeptide. In addition to the autophosphorylated peptide of 25 kDa, a phosphopeptide of 7 kDa was detected in the cyanogen bromide-digested fragments of the c-Src-associated EGF receptor phosphorylated in vitro in an EGF-dependent manner. In phosphopeptide mapping, tryptic digest of the 7-kDa phosphopeptide was shown to co-migrate with that of the phosphorylated Y845 peptide. The 7-kDa phosphopeptide was found to be phosphorylated exclusively on tyrosine. These results suggest that c-Src can phosphorylate EGF receptor on Y845 in an EGF-dependent manner. Furthermore, we confirmed that the same site of the c-Src-associated EGF receptor was phosphorylated in EGF-treated A431 cells.

The adaptor protein Shc was prepared as glutathione S-transferase fusion proteins (GST–Shc) and used as in vitro substrate for c-Src. Since phosphotyrosine-binding domain of Shc has been shown to bind phosphatidyl-inositol 4,5-bisphosphate (PtdIns(4,5)P2) [Zhou et al. (1995) Nature 378, 584–592], effect of PtdIns(4,5)P2 on the phosphorylation of GST–Shc by c-Src was examined. PtdIns(4,5)P2 stimulated the phosphorylation of GST–Shc without any effect on the c-Src activity as judged by both its autophosphorylation and phosphorylation of exogenous substrate, Cdc2 peptide. On the other hand, phosphatidylserine, phosphatidic acid, phosphatidylinositol, and phosphatidylinositol 4-phosphate but not phosphatidylcholine stimulated the c-Src activity itself. Km for GST–Shc in the presence of 1 μM PtdIns(4,5)P2 was calculated to be 90 nM. The PtdIns(4,5)P2-dependent phosphorylation of GST–Shc was inhibited by a GST–fusion protein containing the phosphotyrosine-binding domain of Shc. These results suggest that PtdIns(4,5)P2 can act as a regulator of phosphorylation of Shc by c-Src through its binding to Shc.

We have analyzed tyrosine-phosphorylated proteins in Xenopus laevis eggs before and after fertilization by immunoblotting with anti-phosphotyrosine antibody. A number of egg proteins with different subcellular distribution became tyrosine-phosphorylated or dephosphorylated within 30 min after insemination. Tyrosine kinase-specific inhibitors genistein and herbimycin A were found to inhibit sperm-induced egg activation judged by the egg cortical contraction. Surprisingly, sodium orthovanadate, a tyrosine phosphatase inhibitor, also inhibited the egg activation. Moreover, we found that fertilization-dependent tyrosine dephosphorylation of 42-kDa mitogen-activated protein kinase was inhibited in genistein-treated eggs. These results suggest that both protein-tyrosine phosphorylation and dephosphorylation pathways play an important role in the sperm-induced Xenopus egg activation.

The mechanisms of cell death induced by hypoxia or ischemia are not yet fully understood. We have previously demonstrated that cell death induced by hypoxia occurs independently of caspases, and is mediated by phospholipase A(2) (PLA(2)). Here, we show that p38 mitogen-activated protein kinase is activated under hypoxia. A selective inhibitor of p38 or decrease in the p38alpha protein level prevents hypoxia-induced cell death. The p38 inhibitor abolishes PLA(2) activation by hypoxia, indicating that p38 acts upstream of PLA(2). The antioxidant N-acetyl-cysteine inhibits activation of p38 and cell death induced by hypoxia, indicating that reactive oxygen species (ROS) are responsible for p38 activation. These results demonstrate that the ROS/p38/PLA(2) signaling axis has a crucial role in caspase-independent cell death induced by hypoxia.