Molecular cloning and expression profile of Xenopus calcineurin A subunit(1)

We have cloned a cDNA encoding a catalytic subunit of calcineurin (CnA) expressed in Xenopus oocytes. The deduced amino acid sequence indicates 96.3% and 96.8% identities with the mouse and human CnAalpha isoforms, respectively. Xenopus CnA (XCnA) RNA and protein are expressed as maternal and throughout development. Recombinant XCnA protein interacted with calmodulin in the presence of Ca(2+). Deletion of calmodulin binding domain and auto-inhibitory domain revealed calcium independent phosphatase activity, thereby showing that XCnA is likely to be modulated by both calmodulin and calcium.     

Self-amplification system for recombinant adeno-associated virus production

A recently reported system for recombinant adeno-associated virus (rAAV) production does not require infection of a helper virus and depends on the transfection with a huge amount of three plasmids: AAV-vector, AAV-helper, and adenovirus-helper plasmids. Toward simplifying rAAV production, as a first step, we tested the use of the rAAV itself instead of the AAV-vector plasmid as a source of rAAV DNA and determined the optimal timing of infection and dose of the input rAAV. When 293 cells were infected just after transfection with 100 particles/cell of rAAV, irrespective of the purity, CsCl-purified or crude, up to 2000 particles/cell of rAAV were produced (9- to 20-fold self-amplification), a yield comparable to that obtained by an adenovirus-free transfection. These results indicate that infection of rAAV can greatly reduce the amount of plasmid DNA for a large-scale transfection. This strategy will also be useful when applied to packaging cell lines inducibly expressing Rep and Cap proteins.     

Stimulation of rat hepatocyte proliferation in vitro and in vivo by factors derived from the bovine small intestinal mucosa

Summary A factor with a molecular weight of less than 1 kDa in the mucosa of the bovine small intestine (low molecular weight factor or LMW factor) stimulated DNA synthesis in rat hepatocytes in primary culture. This factor only showed its activity when it was added with a larger factor with a molecular weight of 30 kDa that was also found in the same tissue (high molecular weight factor or HMW factor). The LMW factor probably acts to enhance the action of a hepatotrophic growth factor, since EGF and HGF can substitute for the HMW factor. The action of the LMW factor was not due to the actions of low molecular weight substances such as norepinephrine, estradiol, triiodothyronine, and putrescine, which enhance the action of EGF or HGF, since substantial amounts of these substances were not found in the extract. When intraperitoneally administered into rats, after two-thirds hepatectomy, the LMW factor enhanced hepatocyte proliferation without the administration of the HMW factor. In the regenerating liver, a hepatotrophic growth factor(s), which acts synergistically with the LMW factor, might be properly provided, but the supply of the LMW factor might be below the level that maximally stimulates hepatocyte proliferation.     

Fear Conditioning Induced by Interpersonal Conflicts in Healthy Individuals

Psychophysiological markers have been focused to investigate the psychopathology of psychiatric disorders and personality subtypes. In order to understand neurobiological mechanisms underlying these conditions, fear-conditioning model has been widely used. However, simple aversive stimuli are too simplistic to understand mechanisms because most patients with psychiatric disorders are affected by social stressors. The objective of this study was to test the feasibility of a newly-designed conditioning experiment using a stimulus to cause interpersonal conflicts and examine associations between personality traits and response to that stimulus. Twenty-nine healthy individuals underwent the fear conditioning and extinction experiments in response to three types of stimuli: a simple aversive sound, disgusting pictures, and pictures of an actors’ face with unpleasant verbal messages that were designed to cause interpersonal conflicts. Conditioned response was quantified by the skin conductance response (SCR). Correlations between the SCR changes, and personality traits measured by the Zanarini Rating Scale for Borderline Personality Disorder (ZAN-BPD) and Revised NEO Personality Inventory were explored. The interpersonal conflict stimulus resulted in successful conditioning, which was subsequently extinguished, in a similar manner as the other two stimuli. Moreover, a greater degree of conditioned response to the interpersonal conflict stimulus correlated with a higher ZAN-BPD total score. Fear conditioning and extinction can be successfully achieved, using interpersonal conflicts as a stimulus. Given that conditioned fear caused by the interpersonal conflicts is likely associated with borderline personality traits, this paradigm could contribute to further understanding of underlying mechanisms of interpersonal fear implicated in borderline personality disorder.     

Probucol-Induced α-Tocopherol Deficiency Protects Mice against Malaria Infection

The emergence of malaria pathogens having resistance against antimalarials implies the necessity for the development of new drugs. Recently, we have demonstrated a resistance against malaria infection of α-tocopherol transfer protein knockout mice showing undetectable plasma levels of α-tocopherol, a lipid-soluble antioxidant. However, dietary restriction induced α-tocopherol deficiency is difficult to be applied as a clinical antimalarial therapy. Here, we report on a new strategy to potentially treat malaria by using probucol, a drug that can reduce the plasma α-tocopherol concentration. Probucol pre-treatment for 2 weeks and treatment throughout the infection rescued from death of mice infected with Plasmodium yoelii XL-17 or P. berghei ANKA. In addition, survival was extended when the treatment started immediately after parasite inoculation. The ratio of lipid peroxidation products to parent lipids increased in plasma after 2 weeks treatment of probucol. This indicates that the protective effect of probucol might be mediated by the oxidative stressful environment induced by α-tocopherol deficiency. Probucol in combination with dihydroartemisin suppressed the proliferation of P. yoelii XL-17. These results indicated that probucol might be a candidate for a drug against malaria infection by inducing α-tocopherol deficiency without dietary α-tocopherol restriction.     

Effectiveness of Trivalent Inactivated Influenza Vaccine in Children Estimated by a Test-Negative Case-Control Design Study Based on Influenza Rapid Diagnostic Test Results

We assessed vaccine effectiveness (VE) against medically attended, laboratory-confirmed influenza in children 6 months to 15 years of age in 22 hospitals in Japan during the 2013–14 season. Our study was conducted according to a test-negative case-control design based on influenza rapid diagnostic test (IRDT) results. Outpatients who came to our clinics with a fever of 38°C or over and had undergone an IRDT were enrolled in this study. Patients with positive IRDT results were recorded as cases, and patients with negative results were recorded as controls. Between November 2013 and March 2014, a total of 4727 pediatric patients (6 months to 15 years of age) were enrolled: 876 were positive for influenza A, 66 for A(H1N1)pdm09 and in the other 810 the subtype was unknown; 1405 were positive for influenza B; and 2445 were negative for influenza. Overall VE was 46% (95% confidence interval [CI], 39–52). Adjusted VE against influenza A, influenza A(H1N1)pdm09, and influenza B was 63% (95% CI, 56–69), 77% (95% CI, 59–87), and 26% (95% CI, 14–36), respectively. Influenza vaccine was not effective against either influenza A or influenza B in infants 6 to 11 months of age. Two doses of influenza vaccine provided better protection against influenza A infection than a single dose did. VE against hospitalization influenza A infection was 76%. Influenza vaccine was effective against influenza A, especially against influenza A(H1N1)pdm09, but was much less effective against influenza B.     

TAE226, a Bis-Anilino Pyrimidine Compound,Inhibits the EGFR-Mutant Kinase Including T790M Mutant to Show Anti-Tumor Effect on EGFR-Mutant Non-Small Cell Lung Cancer Cells

TAE226, a bis-anilino pyrimidine compound, has been developed as an inhibitor of focal adhesion kinase (FAK) and insulin-like growth factor-I receptor (IGF-IR). In this study, we investigated the effect of TAE226 on non-small-cell lung cancer (NSCLC), especially focusing on the EGFR mutational status. TAE226 was more effective against cells with mutant EGFR, including the T790M mutant, than against cells with wild-type one. TAE226 preferentially inhibited phospho-EGFR and its downstream signaling mediators in the cells with mutant EGFR than in those with wild-type one. Phosphorylation of FAK and IGF-IR was not inhibited at the concentration at which the proliferation of EGFR-mutant cells was inhibited. Results of the in vitro binding assay indicated significant differences in the affinity for TAE226 between the wild-type and L858R (or delE746_A750) mutant, and the reduced affinity of ATP to the L858R (or delE746_A750) mutant resulted in good responsiveness of the L858R (or delE746_A750) mutant cells to TAE226. Of interest, the L858R/T790M or delE746_A750/T790M mutant enhanced the binding affinity for TAE226 compared with the L858R or delE746_A750 mutant, resulting in the effectiveness of TAE226 against T790M mutant cells despite the T790M mutation restoring the ATP affinity for the mutant EGFR close to that for the wild-type. TAE226 also showed higher affinity of about 15-fold for the L858R/T790M mutant than for the wild-type one by kinetic interaction analysis. The anti-tumor effect against EGFR-mutant tumors including T790M mutation was confirmed in mouse models without any significant toxicity. In summary, we showed that TAE226 inhibited the activation of mutant EGFR and exhibited anti-proliferative activity against NSCLCs carrying EGFR mutations, including T790M mutation.     

Differences in the heat stress associated with white sportswear and being semi-nude in exercising humans under conditions of radiant heat and wind at a wet bulb globe temperature of greater than 28 °C

This study investigated whether wearing common white sportswear can reduce heat stress more than being semi-nude during exercise of different intensities performed under radiant heat and wind conditions, such as a hot summer day. After a 20-min rest period, eight male subjects performed three 20 min sessions of cycling exercise at a load intensity of 20 % or 50 % of their peak oxygen uptake (VO_2peak) in a room maintained at a wet bulb globe temperature (WBGT) of 28.7?±?0.1 °C using two spot lights and a fan (0.8 m/s airflow). Subjects wore common white sportswear (WS) consisting of a long-sleeved shirt (45 % cotton and 55 % polyester) and short pants (100 % polyester), or only swimming pants (SP) under the semi-nude condition. The mean skin temperature \( \left(\overline{T} sk\right) \) was greater when subjects wore SP than WS under both the 20 % and 50 % exercise conditions. During the 50 % exercise, the rating of perceived exertion (RPE) and thermal sensation (TS), and the increases in esophageal temperature (ΔTes) and heart rate were significantly higher (P?<?0.001–0.05), or tended to be higher (P?<?0.07), in the WS than SP trials at the end of the third 20-min exercise session. The total sweat loss (m _sw,tot) was also significantly higher in the WS than in the SP trials (P?<?0.05). However, during the 20 % exercise, the m _sw,tot during exercise, and the ΔTes, RPE and TS at the end of the second and third sessions of exercise did not differ significant between conditions. The heat storage (S), calculated from the changes in the mean body temperature (0.9Tes + 0.1 \( \overline{T} sk \) ), was significantly lower in the WS trials than in the SP trials during the 20 min resting period before exercise session. However, S was similar between conditions during the 20 % exercise, but was greater in the WS than in the SP trials during 50 % exercise. These results suggest that, under conditions of radiant heat and wind at a WBGT greater than 28 °C, the heat stress associated with wearing common WS is similar to that of being semi-nude during light exercise, but was greater during moderate exercise, and the storage of body heat can be reduced by wearing WS during rest periods.