Development and evaluation of human T‐cell leukemia virus‐1 and ‐2 multiplex quantitative PCR

The diagnosis of human T ‐cell leukemia virus type 1 (HTLV‐1) infection in Japan is usually performed by serological testing, but the high rate of indeterminate results from western blotting makes it difficult to assess the infection accurately. Nucleic acid tests for HTLV‐1 and/or HTLV‐2 are used to confirm infection with HTLV‐1 and/or HTLV‐2 and are also used for the follow‐up of HTLV‐1 related diseases. To prepare a highly sensitive method that can discern infection with HTLV‐1 and HTLV‐2, a multiplex quantitative polymerase chain reaction (qPCR) by large‐scale primer screening was developed. Sensitivity and specificity were evaluated by serial dilution of cell lines and by testing with known clinical samples. The resulting multiplex qPCR can detect about four copies of HTLV‐1 provirus per 10⁵ cells. Moreover, HTLV‐1 provirus could be detected in 97.2% (205 of 211) of HTLV‐1 seropositive clinical samples. These sensitivities were sufficiently high compared with the methods reported previously. Also, all the HTLV‐2 seropositive clinical samples tested were found to be positive by this method (three of three). In conclusion, this method can successfully and simultaneously detect both types of HTLV‐1 and HTLV‐2 provirus with extremely high sensitivity.     

Selective antiproliferative activity of caffeic acid phenethyl ester analogues on highly liver-metastatic murine colon 26-L5 carcinoma cell line

Caffeic acid phenethyl ester (CAPE, 2) and its twenty analogues (1, 3-21) were prepared. These esters were tested by MTT assay on growth of murine colon 26-L5 carcinoma, murine B16-BL6 malonoma, murine Lewis lung carcinoma, human HT-1080 fibrosarcoma, human lung A549 adenocarcinoma, and human cervix HeLa adenocarcinoma cell lines. It was found that CAPE analogues possessed selective antiproliferative activity toward highly liver-metastatic murine colon 26-L5 carcinoma cell line. Among them, 4-phenylbutyl caffeate (4), (Z)-8-phenyl-7-octenyl (10a) and (E)-8-phenyl-7-octenyl (10b) caffeate showed the most potent antiproliferative activity (EC50 value, 0.02 microM). In addition, CAPE (2) induced DNA fragmentation at concentrations of 1 to 10 microg/mL towards murine colon 26-L5 carcinoma cells.     

Neuraminidase enhances the initial steps of human T-cell leukemia virus type 1 replication

Human T-cell leukemia virus type 1 (HTLV-1) infection is involved in the development of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. A high HTLV-1 proviral load in circulating lymphocytes of HTLV-1 carriers is a risk factor for HTLV-1-related diseases. The virus–cell interaction is linked to viral tropism and pathogenesis. Characterization of the factors that affect HTLV-1 infection is important for preventing HTLV-1 infection. HTLV-1 virions are believed to be weakly infectious under cell culture conditions; however, we found that the treatment of HTLV-1 virions with microbial neuraminidase, an enzyme catalyzing the removal of sialic acid residues from various glycoconjugates, enhanced the number of proviral DNAs in infected cells in a dose-dependent manner. Neuraminidase treatment of virions, but not target cells, enhanced viral binding and entry into cells and viral infectivity; treatment of target cells prior to infection had no effect. Moreover, the number of HTLV-1-mediated syncytia was higher in the presence of neuraminidase. Our results suggest a possible contribution of microbial agents carrying neuraminidase activity to HTLV-1 pathogenesis.     

Phosphorylation at Tyr-694 of Nogo-A by Src-family kinases

Nogo-A is a neurite outgrowth inhibitor protein associated with myelin in the central nervous system. Unexpectedly, targeted disruption of Nogo-A in mice results in little or no improvement of axonal regeneration, suggesting that Nogo-A has other functions and/or receives complex regulations to exert its inhibitory functions. Here, we have found that Nogo-A becomes phosphorylated at Tyr-694 in the N-terminal region. The phosphorylation is mediated co-operatively by Src-family tyrosine kinases, which play many important roles in the nervous system. Levels of tyrosine phosphorylation of Nogo-A seem to be irrelevant to developmental stages of oligodendrocytes, and might be regulated by specific extracellular stimuli. Identification of tyrosine phosphorylation of Nogo-A will introduce an additional level of complexity into Nogo-A functions.     

NR2B tyrosine phosphorylation modulates fear learning as well as amygdaloid synaptic plasticity

Phosphorylation of neural proteins in response to a diverse array of external stimuli is one of the main mechanisms underlying dynamic changes in neural circuitry. The NR2B subunit of the NMDA receptor is tyrosine-phosphorylated in the brain, with Tyr-1472 its major phosphorylation site. Here, we generate mice with a knockin mutation of the Tyr-1472 site to phenylalanine (Y1472F) and show that Tyr-1472 phosphorylation is essential for fear learning and amygdaloid synaptic plasticity. The knockin mice show impaired fear-related learning and reduced amygdaloid long-term potentiation. NMDA receptor-mediated CaMKII signaling is impaired in YF/YF mice. Electron microscopic analyses reveal that the Y1472F mutant of the NR2B subunit shows improper localization at synapses in the amygdala. We thus identify Tyr-1472 phosphorylation as a key mediator of fear learning and amygdaloid synaptic plasticity.     

Hyperphosphorylation of Tau Induced by Naturally Secreted Amyloid-β at Nanomolar Concentrations Is Modulated by Insulin-dependent Akt-GSK3β Signaling Pathway

Alzheimer disease (AD) is neuropathologically characterized by the formation of senile plaques from amyloid-β (Aβ) and neurofibrillary tangles composed of phosphorylated Tau. Although there is growing evidence for the pathogenic role of soluble Aβ species in AD, the major question of how Aβ induces hyperphosphorylation of Tau remains unanswered. To address this question, we here developed a novel cell coculture system to assess the effect of extracellular Aβ at physiologically relevant levels naturally secreted from donor cells on the phosphorylation of Tau in recipient cells. Using this assay, we demonstrated that physiologically relevant levels of secreted Aβ are sufficient to cause hyperphosphorylation of Tau in recipient N2a cells expressing human Tau and in primary culture neurons. This hyperphosphorylation of Tau is inhibited by blocking Aβ production in donor cells. The expression of familial AD-linked PSEN1 mutants and APP ΔE693 mutant that induce the production of oligomeric Aβ in donor cells results in a similar hyperphosphorylation of Tau in recipient cells. The mechanism underlying the Aβ-induced Tau hyperphosphorylation is mediated by the impaired insulin signal transduction because we demonstrated that the phosphorylation of Akt and GSK3β upon insulin stimulation is less activated under this condition. Treating cells with the insulin-sensitizing drug rosiglitazone, a peroxisome proliferator-activated receptor γ agonist, attenuates the Aβ-dependent hyperphosphorylation of Tau. These findings suggest that the disturbed insulin signaling cascade may be implicated in the pathways through which soluble Aβ induces Tau phosphorylation and further support the notion that correcting insulin signal dysregulation in AD may offer a potential therapeutic approach.     

Arabidopsis sos1 mutant in a salt-tolerant accession revealed an importance of salt acclimation ability in plant salt tolerance

An analysis of the salinity tolerance of 354 Arabidopsis thaliana accessions showed that some accessions were more tolerant to salt shock than the reference accession, Col-0, when transferred from 0 to 225 mM NaCl. In addition, several accessions, including Zu-0, showed marked acquired salt tolerance after exposure to moderate salt stress. It is likely therefore that Arabidopsis plants have at least two types of tolerance, salt shock tolerance and acquired salt tolerance. To evaluate a role of well-known salt shock tolerant gene SOS1 in acquired salt tolerance, we isolated a sos1 mutant from ion-beam-mutagenized Zu-0 seedlings. The mutant showed severe growth inhibition under salt shock stress owing to a single base deletion in the SOS1 gene and was even more salt sensitive than Col-0. Nevertheless, it was able to survive after acclimation on 100 mM NaCl for 7 d followed by 750 mM sorbitol for 20 d, whereas Col-0 became chlorotic under the same conditions. We propose that genes for salt acclimation ability are different from genes for salt shock tolerance and play an important role in the acquisition of salt or osmotic tolerance.     

Regulation of Neurite Growth by Inorganic Pyrophosphatase 1 via JNK Dephosphorylation

Neural cell differentiation during development is controlled by multiple signaling pathways, in which protein phosphorylation and dephosphorylation play an important role. In this study, we examined the role of pyrophosphatase1 (PPA1) in neuronal differentiation using the loss and gain of function analysis. Neuronal differentiation induced by external factors was studied using a mouse neuroblastoma cell line (N1E115). The neuronal like differentiation in N1E115 cells was determined by morphological analysis based on neurite growth length. In order to analyze the loss of the PPA1 function in N1E115, si-RNA specifically targeting PPA1 was generated. To study the effect of PPA1 overexpression, an adenoviral gene vector containing the PPA1 gene was utilized to infect N1E115 cells. To address the need for pyrophosphatase activity in PPA1, D117A PPA1, which has inactive pyrophosphatase, was overexpressed in N1E115 cells. We used valproic acid (VPA) as a neuronal differentiator to examine the effect of PPA1 in actively differentiated N1E115 cells. Si-PPA1 treatment reduced the PPA1 protein level and led to enhanced neurite growth in N1E115 cells. In contrast, PPA1 overexpression suppressed neurite growth in N1E115 cells treated with VPA, whereas this effect was abolished in D117A PPA1. PPA1 knockdown enhanced the JNK phosphorylation level, and PPA1 overexpression suppressed it in N1E115 cells. It seems that recombinant PPA1 can dephosphorylate JNK while no alteration of JNK phosphorylation level was seen after treatment with recombinant PPA1 D117A. Enhanced neurite growth by PPA1 knockdown was also observed in rat cortical neurons. Thus, PPA1 may play a role in neuronal differentiation via JNK dephosphorylation.     

Derivation of iPSCs after Culture of Human Dental Pulp Cells under Defined Conditions

Human dental pulp cells (hDPCs) are a promising resource for regenerative medicine and tissue engineering and can be used for derivation of induced pluripotent stem cells (iPSCs). However, current protocols use reagents of animal origin (mainly fetal bovine serum, FBS) that carry the potential risk of infectious diseases and unwanted immunogenicity. Here, we report a chemically defined protocol to isolate and maintain the growth and differentiation potential of hDPCs. hDPCs cultured under these conditions showed significantly less primary colony formation than those with FBS. Cell culture under stringently defined conditions revealed a donor-dependent growth capacity; however, once established, the differentiation capabilities of the hDPCs were comparable to those observed with FBS. DNA array analyses indicated that the culture conditions robustly altered hDPC gene expression patterns but, more importantly, had little effect on neither pluripotent gene expression nor the efficiency of iPSC induction. The chemically defined culture conditions described herein are not perfect serum replacements, but can be used for the safe establishment of iPSCs and will find utility in applications for cell-based regenerative medicine.