Analysis of Euglena gracilis alpha-, beta- and gamma-tubulin genes: introns and pre-mRNA maturation

We have cloned and analyzed alpha-, beta- and gamma-tubulin genes from Euglena gracilis. The gamma-tubulin genes are 6-10 times longer than the alpha- and beta-tubulin genes, owing to the presence of numerous introns. These introns are all of the conventional type, whereas the alpha- and beta-tubulin genes contain both conventional and non-conventional introns. This is the first time that both types of introns have been found in the same gene. In the E. gracilis genome there are two genes for each tubulin, but the level of gamma-tubulin mRNA is 60 times lower than that of alpha- and beta-tubulin RNAs. The distinctive structure of gamma-tubulin genes prompted us to investigate the maturation of its pre-mRNA. We show that trans-splicing occurs before the cis-splicing of the first intron of the pre-mRNA and that polyadenylation occurs after the cis-splicing of the last intron of the pre-mRNA. We propose that mRNA processing is likely to play a role in regulating the amounts of different tubulins in E. gracilis.     

Conformational analysis of the Sda determinant-containing tetrasaccharide and two mimics in aqueous solution by using 1H NMR ROESY spectroscopy in combination with MD simulations

The conformational behaviour of the spacer-linked synthetic Sd^a tetrasaccharide -d-GalpNAc-(14)-[-Neu5Ac-(23)]--d-Galp-(14)--d-GlcpNAc-(1O)(CH₂)₅NH₂ (1) and the two mimics -d-Galp-(14)-[-Neu5Ac-(23)]--d-Galp-(14)--d-GlcpNAc-(1O)(CH₂)₅NH₂ (2) and -d-GlcpNAc-(14)-[-Neu5Ac-(23)]--d-Galp-(14)--d-GlcpNAc-(1O)(CH₂)₅NH₂ (3) were investigated by ¹H NMR spectroscopy in combination with molecular dynamics (MD) simulations in water. Experimental 2D ¹H ROESY cross-peak intensities (ROEs) of the tetrasaccharides were compared with calculated ROEs derived from MD trajectories using the CROSREL program. Analysis of these data indicated that the oligosaccharidic skeletons of the compounds 1–3 are rather rigid, especially the -d-Hex(NAc)-(14)-[-Neu5Ac-(23)]--d-Galp fragments. The - Neu5-Ac-(23)--d-Galp linkage occurred in two different energy minima in the three-dimensional structure of the compounds 1–3 in aqueous solution. Experimental data and dynamics simulations supported the finding that the higher energy rotamer (CHEAT forcefield) was abundant in compounds 1 and 3 due to the existence of a hydrogen bond between the carboxyl group of the sialic acid and the acetamido group of the terminal monosaccharide (GalNAc or GlcNAc) unit. The conformational similarity between 1 and 3 leads to the suggestion that also their activities will be alike.     

Effects of the N-linked glycans on the 3D structure of the free alpha-subunit of human chorionic gonadotropin

To gain insight into intramolecular carbohydrate-protein interactions at the molecular level, the solution structure of differently deglycosylated variants of the alpha-subunit of human chorionic gonadotropin have been studied by NMR spectroscopy. Significant differences in chemical shifts and NOE intensities were observed for amino acid residues close to the carbohydrate chain at Asn78 upon deglycosylation beyond Asn78-bound GlcNAc. As no straightforward strategy is available for the calculation of the NMR structure of intact glycoproteins, a suitable computational protocol had to be developed. To this end, the X-PLOR carbohydrate force field designed for structure refinement was extended and modified. Furthermore, a computational strategy was devised to facilitate successful protein folding in the presence of extended glycans during the simulation. The values for phi and psi dihedral angles of the glycosidic linkages of the oligosaccharide core fragments GlcNAc2(beta1-4)GlcNAc1 and Man3(beta1-4)GlcNAc2 are restricted to a limited range of the broad conformational energy minima accessible for free glycans. This demonstrates that the protein core affects the dynamic behavior of the glycan at Asn78 by steric hindrance. Reciprocally, the NMR structures indicate that the glycan at Asn78 affects the stability of the protein core. The backbone angular order parameters and displacement data of the generated conformers display especially for the beta-turn 20-23 a decreased structural order upon splitting off the glycan beyond the Asn78-bound GlcNAc. In particular, the Asn-bound GlcNAc shields the protein surface from the hydrophilic environment through interaction with predominantly hydrophobic amino acid residues located in both twisted beta-hairpins consisting of residues 10-28 and 59-84.     

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.