Differentiation of rat pheochromocytoma cells by fomitellic acids, specific DNA polymerase inhibitors

Fomitellic acid (FA) A and B are specific inhibitors of DNA polymerase alpha and beta. They showed cytotoxicity against rat pheochromocytoma cells (PC-12 cells) in a concentration-dependent manner. However, after PC-12 cells were cultivated with low concentrations of FAs, the cells extended neurites in greater degree similar to the cells cultivated with nerve growth factor. Another DNA polymerase alpha inhibitor, aphidicolin, also induced neurite outgrowth. Furthermore, PC-12 cells were strongly immunostained with anti-alpha-tubulin or anti-tau antibody after the treatment with FAs. These results suggest that weak inhibition of DNA polymerase activity induces the neurite outgrowth in PC-12 cells.     

Low-frequency Raman spectra of lysozyme crystals and oriented DNA films: dynamics of crystal water.

We observed low-frequency Raman spectra of tetragonal lysozyme crystals and DNA films, with varying water content of the samples. The spectra are fitted well by sums of relaxation modes and damped harmonic oscillators in the region from approximately 1 cm(-1) to 250 cm(-1). The relaxation modes are due to crystal water, and the distribution of relaxation times is determined. In wet samples, the relaxation time of a small part of the water molecules is a little longer than that of bulk water. The relaxation time of a considerable part of the crystal water, which belongs mainly to the secondary hydration shell, is an order of magnitude longer than that of bulk water. Furthermore, the relaxation time of some water molecules in the primary hydration shell of semidry samples is shorter than we expected. Thus we have shown that low-frequency Raman measurements combined with properly oriented samples can give specific information on the dynamics of hydration water in the ps range. On the other hand, we concluded, based on polarized Raman spectra of lysozyme crystals, that the damped oscillators correspond to essentially intramolecular vibrational modes.     

A Rapid Latex Agglutination Assay for the Detection of Penicillin-Binding Protein 2′

A simple and rapid slide latex agglutination assay was developed to detect penicillin-binding protein 2′ (PBP2′) from isolates of staphylococi. PBP2′ present in the membranes of methicillin-resistant Staphylococcus aureus (MRSA) or methicillin-resistant coagulase negative staphylococci (MRCNS) was rapidly extracted by alkaline treatment and, by combining with a slide agglutination reaction using latex particles sensitized with monoclonal antibodies raised against it, PBP2′ could be detected from a single loopful of cells taken from agar plates not containing beta-lactum antibiotics within 15 min. In a study of clinical isolates previously characterized as either MRSA or methicillin-susceptible Staphylococcus aureus (MSSA) by antibiotic susceptibility testing, 231 specimens of 232 MRSA were PBP2′ positive by latex agglutination, and the 87 specimens of MSSA were all negative. One specimen identified as MRSA by susceptibility testing but PBP2′ negative by latex agglutination was confirmed as mecA gene negative by PCR. This simple and rapid slide latex reagent should be useful in clinical diagnostics.     

Augmentation of the antitumor effect of adoptive immunotherapy by in vivo sensitization of EL-4 lymphoma and pre-treatment with sizofiran

The antitumor effects of adoptive immunotherapy using LAKcells treated with sizofiran (SPG) following in vivoantigen sensitization with EL-4 lymphoma (EsLAK),comparing nonsensitized LAK cells (sLAK), were studied inmice with intraperitoneal implantation of EL-4 lymphoma.EL-4 cells treated with Mitomycin C (100 g /ml) wereintroduced by inoculation into the peritoneum of C57BL/6mice for antigen sensitization. Four days later, SPG (100g) was intramuscularly injected. Three days after SPG administration, mononuclear cells obtained from the spleen were prepared for LAK cells (EsLAK). The following resultswere obtained: 1) The survival period was significantlygreater in the sLAK and EsLAK groups than in the controlgroup. The survival period in the EsLAK group wassignificantly greater than that in the sLAK group. 2) Thenumber of EL-4 cells in the peritoneal exudate cells 11days postimplantation was lowest in the EsLAK group, andthe number of lymphocytes including LGL was largest in theEsLAK group, compared with the sLAK group and the controlgroup. 3 ) The EsLAK cells showed significantly moreenhanced cytotoxic activity against EL-4 than the sLAKcells. 4) Histopathological findingsof metastatic lesions of the liver and spleen stained by HE11 days postimplantation showed less infiltrating tumorcells and more lymphocytic infiltrations in the sLAK andEsLAK groups compared with the control group. Theseresults suggest that induction of LAK cells byadministration of SPG to lymphocytes treated by in vivosensitization with tumor antigen increasesthe efficacy of adoptive immunotherapy.     

A Marked Increase in Free Copper Levels in the Plasma and Liver of LEC Rats: An Animal Model for Wilson Disease and Liver Cancer

Most of copper present in rat plasma and liver binds to caeruloplasmin and metallothionein, respectively, and is not redox active. However, free forms of copper including loosely bound forms to other molecules are redox active. We assessed the free copper in Long-Evans rats with a cinnamon-like coat color (LEC rats), an animal model of Wilson disease and liver cancer. Compared to those of control rats, the liver and plasma of LEC rats showed a marked elevation of free copper, especially at the stage of acute hepatitis, in parallel with an increase of total copper levels in the livers and a decrease of plasma caeruloplasmin (ferroxidase I) activity. At the onset of jaundice, the total copper levels, however, decreased in liver, but increased in plasma, while free copper levels in both liver and plasma remained higher. Free iron levels in both liver and plasma were also determined and did not change significantly, except for the case of plasma in jaundiced rats. The data are consistent with a proposal in which increased levels of redox active free copper in the liver of LEC rats catalyze Fenton-type reactions, producing a large flux of hydroxyl radicals that would play an important role in the observed liver dysfunction, leading to acute hepatitis, and, finally, hepatocarcinoma. This is the first demonstration that the free copper may participate in the pathophysiology of the LEC rats and Wilson disease.     

Host range expansion by Rhopalomyia yomogicola (Diptera: Cecidomyiidae) from a native to an alien species of Artemisia (Asteraceae) in Japan

In 2001, subconical galls were found on the leaves of an alien Artemisia species (Asteraceae) in Ibusuki City, Kagoshima Prefecture, Japan. These galls were quite similar to those induced by Rhopalomyia yomogicola (Diptera: Cecidomyiidae) on Artemisia princeps, Artemisia montana, and Artemisia japonica in Japan. The morphological features of the pupal head and molecular sequencing data indicated that the gall midge from the alien Artemisia was identical to R. yomogicola. Usually, galling insects do not expand readily their host range to alien plants, but R. yomogicola is considered to have expanded its host range to the alien Artemisia by its multivoltine life history trait and oligophagous habit across two different botanical sections of the genus Artemisia. Adult abdominal tergites and sternites and immature stages of R. yomogicola are described for the first time and detailed biological information is presented.     

Involvement of IL-1 in the Maintenance of Masseter Muscle Activity and Glucose Homeostasis

Physical exercise reportedly stimulates IL-1 production within working skeletal muscles, but its physiological significance remains unknown due to the existence of two distinct IL-1 isoforms, IL-1α and IL-1β. The regulatory complexities of these two isoforms, in terms of which cells in muscles produce them and their distinct/redundant biological actions, have yet to be elucidated. Taking advantage of our masticatory behavior (Restrained/Gnawing) model, we herein show that IL-1α/1β-double-knockout (IL-1-KO) mice exhibit compromised masseter muscle (MM) activity which is at least partially attributable to abnormalities of glucose handling (rapid glycogen depletion along with impaired glucose uptake) and dysfunction of IL-6 upregulation in working MMs. In wild-type mice, masticatory behavior clearly increased IL-1β mRNA expression but no incremental protein abundance was detectable in whole MM homogenates, whereas immunohistochemical staining analysis revealed that both IL-1α- and IL-1β-immunopositive cells were recruited around blood vessels in the perimysium of MMs after masticatory behavior. In addition to the aforementioned phenotype of IL-1-KO mice, we found the IL-6 mRNA and protein levels in MMs after masticatory behavior to be significantly lower in IL-1-KO than in WT. Thus, our findings confirm that the locally-increased IL-1 elicited by masticatory behavior, although present small in amounts, contributes to supporting MM activity by maintaining normal glucose homeostasis in these muscles. Our data also underscore the importance of IL-1-mediated local interplay between autocrine myokines including IL-6 and paracrine cytokines in active skeletal muscles. This interplay is directly involved in MM performance and fatigability, perhaps mediated through maintaining muscular glucose homeostasis.     

Comparison of the Japanese Orthopaedic Association (JOA) Score and Modified JOA (mJOA) Score for the Assessment of Cervical Myelopathy: A Multicenter Observational Study

Objectives

The Japanese Orthopaedic Association (JOA) score is widely used to assess the severity of clinical symptoms in patients with cervical compressive myelopathy, particularly in East Asian countries. In contrast, modified versions of the JOA score are currently accepted as the standard tool for assessment in Western countries. The objective of the present study is to compare these scales and clarify their differences and interchangeability and verify their validity by comparing them to other outcome measures.

Materials and Methods

Five institutions participated in this prospective multicenter observational study. The JOA and modified JOA (mJOA) proposed by Benzel were recorded preoperatively and at three months postoperatively in patients with cervical compressive myelopathy who underwent decompression surgery. Patient reported outcome (PRO) measures, including Japanese Orthopaedic Association Cervical Myelopathy Evaluation Questionnaire (JOACMEQ), the Short Form-12 (SF-12) and the Neck Disability Index (NDI), were also recorded. The preoperative JOA score and mJOA score were compared to each other and the PRO values. A Bland-Altman analysis was performed to investigate their limits of agreement.

Results

A total of ninety-two patients were included. The correlation coefficient (Spearman’s rho) between the JOA and mJOA was 0.87. In contrast, the correlations between JOA/mJOA and the other PRO values were moderate (|rho| = 0.03 – 0.51). The correlation coefficient of the recovery rate between the JOA and mJOA was 0.75. The Bland-Altman analyses showed that limits of agreement were 3.6 to -1.2 for the total score, and 55.1% to -68.8% for the recovery rates.

Conclusions

In the present study, the JOA score and the mJOA score showed good correlation with each other in terms of their total scores and recovery rates. Previous studies using the JOA can be interpreted based on the mJOA; however it is not ideal to use them interchangeably. The validity of both scores was demonstrated by comparing these values to the PRO values.     

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.     

Protracted Administration of L-Asparaginase in Maintenance Phase Is the Risk Factor for Hyperglycemia in Older Patients with Pediatric Acute Lymphoblastic Leukemia

Although L-asparaginase related hyperglycemia is well known adverse event, it is not studied whether the profile of this adverse event is affected by intensification of L-asparaginase administration. Here, we analyzed the profile of L-asparaginase related hyperglycemia in a 1,176 patients with pediatric acute lymphoblastic leukemia treated according to the Japan Association of Childhood Leukemia Study ALL-02 protocol using protracted L-asparaginase administration in maintenance phase. We determined that a total of 75 L-asparaginase related hyperglycemia events occurred in 69 patients. Although 17 events (17/1176, 1.4%) developed in induction phase, which was lower incidence than those (10–15%) in previous reports, 45 events developed during the maintenance phase with protracted L-asparaginase administration. Multivariate analysis showed that older age at onset (≥10 years) was a sole independent risk factor for L-asparaginase-related hyperglycemia (P<0.01), especially in maintenance phase. Contrary to the previous reports, obesity was not associated with L-asparaginase-related hyperglycemia. These findings suggest that protracted administration of L-asparaginase is the risk factor for hyperglycemia when treating adolescent and young adult acute lymphoblastic leukemia patients.