HSP70 expression in the CNS in response to exercise and heat stress in rats

We havepreviously documented the regional distribution of 70-kDa heat shockprotein (HSP70) in brains of rats made hyperthermic by brief exposureto high-powered microwaves (HPM; 2.06 GHz). We now compare HSP70expression induced by HPM exposure to that induced by exertionaland/or environmental heat stress. Rats were chronicallyimplanted with a temperature probe guide in the hypothalamic region ofthe brain (T_br). After recovery,the following treatment groups were examined: HPM; sham exposed;treadmill exercise at room temperature (24°C; Ex-1); treadmillexercise in a warm environment (34°C; Ex-2); and sedentary groups(Sed-1 and Sed-2), in which ambient temperature was adjusted so thatthe T_br mimicked the T_br in the corresponding exercisegroups. Significant HSP70 expression occurred only in the hyperthermic(Ex-2, Sed-2, and HPM) groups. The pattern of HSP70 expression wassimilar among Ex-2 and Sed-2 rats but differed from that in HPM rats.We conclude that 1) the pattern ofHSP70 expression differs between HPM and nonmicrowave heating, and2) exercise alone was not sufficientto induce central HSP70 expression.

     

Journal Impact Factor Shapes Scientists’ Reward Signal in the Prospect of Publication

The incentive structure of a scientist’s life is increasingly mimicking economic principles. While intensely criticized, the journal impact factor (JIF) has taken a role as the new currency for scientists. Successful goal-directed behavior in academia thus requires knowledge about the JIF. Using functional neuroimaging we examined how the JIF, as a powerful incentive in academia, has shaped the behavior of scientists and the reward signal in the striatum. We demonstrate that the reward signal in the nucleus accumbens increases with higher JIF during the anticipation of a publication and found a positive correlation with the personal publication record (pJIF) supporting the notion that scientists have incorporated the predominant reward principle of the scientific community in their reward system. The implications of this behavioral adaptation within the ecological niche of the scientist’s habitat remain unknown, but may also have effects which were not intended by the community.     

Functional Significance of Labellum Pattern Variation in a Sexually Deceptive Orchid (Ophrys heldreichii): Evidence of Individual Signature Learning Effects

Mimicking female insects to attract male pollinators is an important strategy in sexually deceptive orchids of the genus Ophrys, and some species possess flowers with conspicuous labellum patterns. The function of the variation of the patterns remains unresolved, with suggestions that these enhance pollinator communication. We investigated the possible function of the labellum pattern in Ophrys heldreichii, an orchid species in which the conspicuous and complex labellum pattern contrasts with a dark background. The orchid is pollinated exclusively by males of the solitary bee, Eucera berlandi. Comparisons of labellum patterns revealed that patterns within inflorescences are more similar than those of other conspecific plants. Field observations showed that the males approach at a great speed and directly land on flowers, but after an unsuccessful copulation attempt, bees hover close and visually scan the labellum pattern for up to a minute. Learning experiments conducted with honeybees as an accessible model of bee vision demonstrated that labellum patterns of different plants can be reliably learnt; in contrast, patterns of flowers from the same inflorescence could not be discriminated. These results support the hypothesis that variable labellum patterns in O. heldreichii are involved in flower-pollinator communication which would likely help these plants to avoid geitonogamy.     

Effects of insulin-like growth factor 1 on muscle atrophy and motor function in rats with brain ischemia

Although insulin-like growth factor 1 (IGF 1) has been used in immobilizated muscles to prevent muscle atrophy, its effects on muscle atrophy after brain ischemia are not known. This study aimed to determine the effects of IGF 1 on preventing muscle atrophy in rats with brain ischemia. Middle cerebral artery occlusion (MCAO) was used to induce the brain ischemia. In the first part of the study, rats were assigned to sham control, ischemic control, and ischemia with different dosages of IGF 1 injection groups to determine the optimal dosage of IGF 1 on preventing muscle atrophy after brain ischemia. In the second part of the study, rats were assigned to sham control, ischemic control, ischemia with IGF 1, or with IGF 1 receptor inhibitor (AG1024) injection groups to determine the specificity of IGF 1 on preventing muscle atrophy after brain ischemia. IGF 1 or AG1024 was injected locally to calf muscles and anterior tibialis (TA) starting from one day after brain ischemia and injections were carried out every other day for 4 times. Muscle weight and myosin heavy chain (MHC) expression in both red (red gastrocnemius and soleus) and white (white gastrocnemius and TA) muscles were significantly decreased after brain ischemia. With at least moderate-dosage (200 ng/100 microl PBS) IGF 1 injection, the muscle weight and MHC protein could be restored in both red and white muscles resulting in better motor performance. However, the high-dose injection of IGF 1 (400 ng/100 microl PBS) did not result in further effects. IGF 1 increased the expression of p-Akt, but such effects were prevented by AG1024 resulting in muscle atrophy and poor motor function. In conclusion, peripheral application of IGF 1 not only prevented muscle atrophy but also enhanced motor function in rats with brain ischemia. The IGF 1-induced PI3K/Akt pathways are important for preventing muscle atrophy induced by brain ischemia.     

A high-throughput, homogeneous, fluorescence polarization assay for inhibitors of hedgehog protein autoprocessing

Hedgehog (Hh) signaling plays an important role in embryonic patterning and adult stem cell renewal but has recently been found also to be involved in certain stem cell cancers. One of the first steps in Hh signaling is the autoprocessing of Hh protein, in which the C-terminal domain (Hh-C) catalyzes a cholesterol-dependent autocleavage reaction that leads to the production of the cholesterol ester of the N-terminal Hh domain (Hh-N), thereby yielding a signaling molecule that activates the Hh pathway by binding to the Patched receptor. This article describes an in vitro, homogeneous assay system that measures changes in fluorescence polarization that accompany the cholesterol-dependent autocleavage of Hh protein. The assay system makes use of a modified Hh protein in which Hh-N, which is not essential for autocleavage, is replaced by a 25-residue peptide containing a tetracysteine motif, complexed with a bisarsenical fluorophore. The assay is quite robust and easily adapted to high-throughput screening in 384-well plates with Z' factors above 0.8. It has been used to screen the National Institutes of Health Clinical Collection, which has led to the identification of 2 compounds that inhibit the cholesterol-dependent autocleavage of Hh protein at micromolar concentrations.     

Effect of hypoxia on the release of vascular endothelial growth factor and testosterone in mouse TM3 Leydig cells

Hypoxia has been shown to stimulate the expression of vascular endothelial growth factor (VEGF), which is a major mediator for angiogenesis and vasculogenesis. During hypoxia, VEGF promotes angiogenesis in the testis. However, the effect of VEGF on the steroidogenesis of testosterone and the cell proliferation in Leydig cells is unclear. To assess the effects and the action mechanisms of hypoxia, a mouse TM3 Leydig cell line was employed in the present study. The Leydig cells were incubated in an incubator chamber (95% N2-5% CO2) for 1-24 h. The cultured media were collected and assayed by testosterone RIA and VEGF enzyme immunoassay. 3-(4,50-Dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide assay was used to detect the proliferation of Leydig cells. The present results showed that the proliferation of Leydig cells was enhanced significantly by hypoxia. The basal VEGF release was increased, and the response of VEGF production to human chorionic gonadotropin (hCG) was also enhanced in hypoxic condition. During hypoxia, administration of hCG or VEGF stimulated proliferation of Leydig cells, but the stimulatory effect was abolished by the administration of anti-VEGF antibody. Higher doses of VEGF stimulated testosterone release in a dose-dependent manner. Administration of anti-VEGF antibody abolished the stimulatory effect of VEGF on testosterone release. These data suggest that hypoxia stimulates cell proliferation and testosterone release in Leydig cells via an increase of VEGF production.     

Deinococcus glutaminyl-tRNA synthetase is a chimer between proteins from an ancient and the modern pathways of aminoacyl-tRNA formation

Glutaminyl-tRNA synthetase from Deinococcus radiodurans possesses a C-terminal extension of 215 residues appending the anticodon-binding domain. This domain constitutes a paralog of the Yqey protein present in various organisms and part of it is present in the C-terminal end of the GatB subunit of GatCAB, a partner of the indirect pathway of Gln-tRNA^Gln formation. To analyze the peculiarities of the structure–function relationship of this GlnRS related to the Yqey domain, a structure of the protein was solved from crystals diffracting at 2.3Å and a docking model of the synthetase complexed to tRNA^Gln constructed. The comparison of the modeled complex with the structure of the E. coli complex reveals that all residues of E. coli GlnRS contacting tRNA^Gln are conserved in D. radiodurans GlnRS, leaving the functional role of the Yqey domain puzzling. Kinetic investigations and tRNA-binding experiments of full length and Yqey-truncated GlnRSs reveal that the Yqey domain is involved in tRNA^Gln recognition. They demonstrate that Yqey plays the role of an affinity-enhancer of GlnRS for tRNA^Gln acting only in cis. However, the presence of Yqey in free state in organisms lacking GlnRS, suggests that this domain may exert additional cellular functions.     

Anti-proliferative effects of evodiamine on human prostate cancer cell lines DU145 and PC3

Prostate carcinoma is one of the most common malignant tumors and has become a more common cancer in men. Previous studies demonstrated that evodiamine (EVO) exhibited anti-tumor activities on several cancers, but its effects on androgen-independent prostate cancer are unclear. In the present study, the action mechanisms of EVO on the growth of androgen-independent prostate cancer cells (DU145 and PC3 cells) were explored. EVO dramatically inhibited the growth and elevated cytotoxicity of DU145 and PC3 cells. The flow cytometric analysis of EVO-treated cells indicated a block of G2/M phase and an elevated level of DNA fragmentation. The G2/M arrest was accompanied by elevated Cdc2 kinase activity, an increase in expression of cyclin B1 and phosphorylated Cdc2 (Thr 161), and a decrease in expression of phosphorylated Cdc2 (Tyr 15), Myt-1, and interphase Cdc25C. TUNEL examination showed that EVO-induced apoptosis was observed at 72 h. EVO elevated the activities of caspase 3, 8, and 9 in DU145 cells, while in PC3 cells only the activities of caspase 3 and 9 were elevated. EVO also triggered the processing of caspase 3 and 9 in both DU145 and PC3 cells. We demonstrate that roscovitine treatment result in the reversion of G2/M arrest in response to EVO in both DU145 and PC3. However, inhibitory effect of roscovitine on EVO-induced apoptosis could only be observed in DU145 rather than PC3. In DU145, G2/M arrest might be a signal for initiation of EVO-triggered apoptosis. Whereas EVO-triggered PC3 apoptosis might be independent of G2/M arrest. These results suggested that EVO inhibited the growth of prostate cancer cell lines, DU145 and PC3, through an accumulation at G2/M phase and an induction of apoptosis.     

Actin deficiency induces cofilin phosphorylation: proteome analysis of HeLa cells after beta-actin gene silencing

Actin-binding proteins regulate the dynamic structure and function of actin filaments in the cell. Much is known about how manipulation of the actin-binding proteins affects the structure and function of actin filaments; however, little is known about how manipulation of actin in the cell affects actin-binding proteins. We addressed this question by utilizing two technologies: RNA interference and 2-dimensional gel electrophoresis. We knocked down beta-actin expression in HeLa cells using short interfering RNA and applied 2-DGE to examine alterations in the HeLa cell proteome. We revealed a 2-5 fold increases of four protein spots on 2-D gels and identified these proteins by mass spectrometry. Three of the four proteins were actin-binding proteins, including cofilin, which promotes both disassembly and assembly of actin filaments but becomes inactivated when phosphorylated. Further examination revealed that the cofilin total protein level barely increased, but the phosphorylated cofilin level increased dramatically in HeLa cells after beta-actin siRNA treatment. These results suggest that in response to siRNA-induced beta-actin deficiency HeLa cells inactivate cofilin by phosphorylation rather than down-regulate its protein expression level. This study also demonstrates that the combination of RNA interference and 2-dimensional gel electrophoresis technologies provides a valuable method to study protein interactions in a specific cellular pathway.