Aryl hydrocarbon receptor signaling involved in the invasiveness of LNCaP cells

There is now mounting evidence that the aryl hydrocarbon receptor (AhR) plays an important role in physiologic responses such as development, cell cycle regulation, immune function and also malignant transformation in various tissues. The strong nuclear AhR expression is observed in the invasive phenotype, and an elevated nuclear AhR expression is associated with a poor prognosis of human prostate cancer. On the other hand, there are conflicting results that the AhR deficiency results in increased susceptibility to prostate tumors in mouse model. In the present study, we investigated AhR expression and its role in the growth and invasiveness of human prostate cancer cells. The AhR protein expression was detected in prostate cancer cell lines and human prostate cancer tissues. A small interfering RNA targeting AhR, constitutive active AhR expression vector, and AhR agonist and antagonist were used to moderate its expression and signaling. The induction of AhR signaling attenuated invasiveness of prostate cancer cells without affecting the cellular growth rate. These results suggest that AhR signaling in prostate cancer cells facilitates invasion of these cells, and modulation with this signaling can be a potential therapeutic target of invasive tumors.     

Local and chemical distribution of phlorotannins in brown algae

The local and chemical distribution of phlorotannins among the Japanese Laminariaceae, Eisenia bicyclis, Ecklonia cava and Ecklonia kurome, was investigated. As a result of light microscopy observations with vanillin-HCl staining, phlorotannins were found to be accumulated within the vegetative cells of the outer cortical layer of the thalli, regardless of the species, stage of growth or organ. Crude phlorotannins comprised about 3.0% of the algal powder for each of the algae. High-performance liquid chromatography (HPLC) showed that the phlorotannins of E. bicyclis were composed of phloroglucinol (0.9%), phloroglucinol tetramer (4.4%), eckol (7.5%), phlorofucofuroeckol A (21.9%), dieckol (23.4%), and 8,8'-bieckol (24.6%), plus some other unknown phenolic compounds (17.3%). The composition of the phlorotannins differed little among the Laminariaceae, except for a significantly larger amount of the tetramer, MW 478, in E. bicyclis.     

Hes genes regulate size, shape and histogenesis of the nervous system by control of the timing of neural stem cell differentiation

Radial glial cells derive from neuroepithelial cells, and both cell types are identified as neural stem cells. Neural stem cells are known to change their competency over time during development: they initially undergo self-renewal only and then give rise to neurons first and glial cells later. Maintenance of neural stem cells until late stages is thus believed to be essential for generation of cells in correct numbers and diverse types, but little is known about how the timing of cell differentiation is regulated and how its deregulation influences brain organogenesis. Here, we report that inactivation of Hes1 and Hes5, known Notch effectors, and additional inactivation of Hes3 extensively accelerate cell differentiation and cause a wide range of defects in brain formation. In Hes-deficient embryos, initially formed neuroepithelial cells are not properly maintained, and radial glial cells are prematurely differentiated into neurons and depleted without generation of late-born cells. Furthermore, loss of radial glia disrupts the inner and outer barriers of the neural tube, disorganizing the histogenesis. In addition, the forebrain lacks the optic vesicles and the ganglionic eminences. Thus, Hes genes are essential for generation of brain structures of appropriate size, shape and cell arrangement by controlling the timing of cell differentiation. Our data also indicate that embryonic neural stem cells change their characters over time in the following order: Hes-independent neuroepithelial cells, transitory Hes-dependent neuroepithelial cells and Hes-dependent radial glial cells.     

Cigarette smoking during pregnancy lowers aromatase cytochrome P-450 in the human placenta

To clarify whether cigarette smoking during pregnancy causes an organic alteration in placental estrogen producing ability, we determined the catalytic activity of aromatase by the tritiated water assay, and tissue level of aromatase cytochrome P-450 (P-450_arom) by the specific enzyme-linked immunosorbent assay, in placental samples from nonsmokers and smokers. As pregnancy progressed, both aromatase activity and P-450_arom concentration increased in placentas from nonsmokers and smokers. However, the gradient of the increase was significantly less in heavy smokers (20 cigarettes a day) than in normal and moderate smokers (<20 cigarettes a day). At term, the mean aromatase activity and P-450_arom concentration in placentas from heavy smokers were significantly lower than in nonsmokers and moderate smokers, while aromatase activity per P-450_arom (turnover rate) and the mean placental weight were comparable among the three groups. In contrast, the ratio of aryl hydrocarbon hydroxylase activity to aromatase activity was higher in placentas from heavy smokers. Immunohistochemical studies showed that P-450_arom was localized in the cytoplasm of syncytiotrophoblasts of chorionic villi in placentas from both nonsmokers and smokers. These results suggest that the induction of placental P-450_arom during gestation is suppressed by maternal smoking, resulting in a reduction in estrogen producing ability, while placental xenobiotic P-450 is induced.     

Fiber types and some contractile properties of extensor digitorum longus and soleus muscles in different animal species

We studied the fiber types and contractile properties of the extensor digitorum longus (EDL) and soleus (SOL) muscles from young adult mice, rats and guinea pigs, and the correlation between these two parameters. Individual fibers in both muscles were classified as fast-twitch glycolytic (FG), fast-twitch oxidative glycolytic (FOG) or slow-twitch oxidative (SO) fibers according to Peter et al., and type II B, II A, or I fibers according to Brooke & Kaiser. Contractile properties were measured in situ at 37 degrees C. The isometric twitch contraction time (CT) and one-half relaxation time (1/2 RT) tended to be shortened in proportion to the area occupied by type II fibers, and type II B fibers. However, the differences between CT and fiber types were not always uniform among the three species. The CT of the rat EDL, in spite of its higher proportion of type II B fibers about 10% was the same as that of the guinea-pig EDL. The SOL of the mouse, composed of about 50% type I (SO) fibers, had a CT about as short as that of the EDL. In the case of the classification by Peter et al., the relationship between the percentage of subgroups of fast-twitch fibers and the CT or 1/2 RT, but not the resistance to fatigue, was not obvious. The resistance to fatigue tended to be enhanced in proportion to the area occupied by FOG in the EDL and by SO (type I) in the SOL. These results suggest that the contractile properties of individual fibers identified histochemically are distinct among animal species, producing interspecies differences in fiber types along with different contractile properties. However, it may be possible to compare the difference between fiber types and CT or 1/2 RT in the classification based on the pH lability of myosin ATPase, and also the difference between fiber types and resistance to fatigue in the classification based on the oxidative enzyme.     

High Rate of Awarding Compensation for Claims of Injuries Related to Clinical Trials by Pharmaceutical Companies in Japan: A Questionnaire Survey

Introduction

International norms and ethical standards have suggested that compensation for research-related injury should be provided to injured research volunteers. However, statistical data of incidence of compensation claims and the rate of awarding them have been rarely reported.

Method

Questionnaire surveys were sent to pharmaceutical companies and medical institutions, focusing on industry-initiated clinical trials aiming at new drug applications (NDAs) on patient volunteers in Japan.

Results

With the answers from pharmaceutical companies, the incidence of compensation was 0.8%, including 0.06% of monetary compensation. Of the cases of compensation claims, 99% were awarded. In turn, with the answers from medical institutions, the incidence of compensation was 0.6%, including 0.4% of serious but not death cases, and 0.04% of death cases. Furthermore, most claims for compensation were initiated by medical institutions, rather than by the patients. On the other hand, with the answers from clinical trial volunteers, 3% of respondents received compensations. These compensated cases were 25% of the injuries which cannot be ruled out from the scope of compensation.

Conclusion

Our study results demonstrated that Japanese pharmaceutical companies have provided a high rate of compensation for clinical trial-related injuries despite the possibility of overestimation. In the era of global clinical development, our study indicates the importance of further surveys to find each country''s compensation policy by determining how it is being implemented based on a survey of the actual status of compensation coming from statistical data.     

Improvement of the post-thaw qualities of Okinawan native Agu pig sperm frozen in an extender supplemented with antiapoptotic PTD-FNK protein

The technical establishment of boar sperm cryopreservation is indispensable for effective breeding of the scarce Okinawan native Agu pig. The objective was to determine whether an artificial anticell death protein (PTD-FNK protein) was capable of improving the quality of cryopreserved Agu sperm. Ejaculated Agu sperm frozen in an extender supplemented with 0, 100, 200, 300, or 400 nm PTD-FNK protein was thawed, and mitochondrial integrity and other sperm characteristics were evaluated. Treatment with 300 nm PTD-FNK protein had the most beneficial effect (P < 0.05) on mitochondrial integrity (45-59%) and sperm motility (56-67%) after freezing-thawing. In particular, the proportion of post-thaw sperm with activated caspase-9 and -3 but not caspase-8 was markedly reduced among sperm frozen in the presence of PTD-FNK protein (P < 0.05), implying protection against apoptotic-cell death in response to mitochondrial damage. There were high levels of intracellular ATP (9.4-10.5 nmol/10⁸ sperm) in post-thaw sperm treated with PTD-FNK protein, and the inhibitory effect of PTD-FNK protein on activation of caspases influenced the increase in the number of sperm with intact DNA (36-53%; P < 0.05). Furthermore, the addition of PTD-FNK protein to the freezing extender strongly preserved the ability of the sperm to penetrate to mature oocytes in all individuals (60-80%; P < 0.05). In conclusion, treatment with PTD-FNK protein in the freezing extender effectively improved post-thaw qualities of fragile Agu sperm through prevention of mitochondrial dysfunction leading to apoptotic-cell death during cryopreservation.     

Developmental changes in the expression pattern of the JNK activator kinase MUK/DLK/ZPK and active JNK in the mouse cerebellum

JNK is one of the key molecules regulating cell differentiation and migration in a variety of cell types, including cerebral cortical neurons. MUK/DLK/ZPK belongs to the MAP kinase-kinase-kinase class of protein kinases for the JNK pathway and is expressed predominantly in neural tissue. We have determined the expression pattern of MUK/DLK/ZPK and active JNK in the cerebellum at different stages of postnatal development. Quantitative analysis by Western blotting has showed that high expression levels of MUK/DLK/ZPK and active JNK are maintained during the postnatal development of the cerebellum, and that these levels decrease in the adult cerebellum. Immunohistochemical staining has revealed, however, that their distribution in the developing cerebellum is considerably different. Although active JNK is highly concentrated in the premigratory zone of the external germinal layer (EGL), high expression of MUK/DLK/ZPK has been observed in the molecular layer and in the premigratory zone. Neither the active JNK nor MUK protein has been detected in the proliferative zone of the EGL. These observations suggest that during the postnatal development of the cerebellum, the MUK-JNK signaling pathway contributes to the regulation of granule cell differentiation and migration; further, the activity of MUK/DLK/ZPK is tightly regulated by posttranslational mechanisms and by its expression level.This work was supported by a Ishizu Shun memorial scholarship and grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan.     

Biological safety in virological field with special considerations on class II biological safety cabinets

The most critical point for the biosafety is not sophisticated devices or facilities, but education of workers and their compliance to the regulation. Appropriate devices should be carefully selected in the introduction of new devices, and they should be properly maintained. The class II biosafety cabinet is one of the delicate safety equipments. It should be kept adequately maintained throughout the lifetime of the cabinet to insure safety of the laboratory. For the maintenance, appropriate measuring equipments should be used by trained technicians. The recently enforced law for control of recombinant DNA researches should be applied for the handling of pathogens even in non-recombinant DNA researches after proper modifications.