Effect of aliphatic alcohols on the conformation of poly (l-ornithine hydrobromide) as studied by square-pulse and reversing-pulse electric birefringence

The electric birefringence and circular dichroism spectra of poly(l-ornithine hydrobromide) have been measured in ethanol/water, 2-propanol/water and tertiary butyl alcohol/water mixtures of various compositions. This charged polypeptide underwent a transition from the coil conformation to the helical conformation at high alcohol content in every case tested. Anomalous birefringence signals, indicative of a field-induced helix-to-coil transition. were observed at high electric fields only in the case of ethanol/water mixtures. The reversing-pulse electric birefringence of this polypeptide has been studied in ethanol/water mixtures and in neutral aqueous solution. Upon rapid reversal of the pulse field, no transient could be observed. This confirms that the electric-field orientation of poly(l-ornithine hydrobromide) results predominantly from the contribution of the counterion-induced dipole moment, regardless of its molecular conformations. It is very probable that the backbone permanent dipole moment of the helical conformation is largely suppressed by the counterion-induced dipole moment in the ionized form.     

Electric field-induced conformational change of poly(L-lysine) studied by transient electric birefringence

Transient electric birefringence measurements on poly(L -lysine hydrobromide) in methanol–water mixtures have been carried out at various solvent compositions in the vicinity of the helix–coil transition region (from 87 to 98 vol % methanol). Anomalous birefringence transients were observed between 90 and 95 vol % methanol above a threshold field strength. A distinct difference between the responses to weak and strong electric fields was noticed over a narrow range of the solvent composition. The effects of polymer concentration and temperature on the field-strength dependence of the birefringence were studied at a solvent composition of 90 vol % methanol where the anomalous transients appeared most clearly. The double logarithmic plots of the steady-state specific birefringence versus the square of field strength for different concentrations and temperatures could be superimposed by shifting them horizontally along the abscissa. The threshold field strength which was determined from the shift factor decreased with decreasing concentration. The results provide further evidence that strong electric fields can cause a helix–coil transition in this system under favorable conditions.     

The RNA-binding protein Puf5 and the HMGB protein Ixr1 contribute to cell cycle progression through the regulation of cell cycle-specific expression of CLB1 in Saccharomyces cerevisiae

Puf5, a Puf-family RNA-binding protein, binds to 3´ untranslated region of target mRNAs and negatively regulates their expression in Saccharomyces cerevisiae. The puf5Δ mutant shows pleiotropic phenotypes including a weakened cell wall, a temperature-sensitive growth, and a shorter lifespan. To further analyze a role of Puf5 in cell growth, we searched for a multicopy suppressor of the temperature-sensitive growth of the puf5Δ mutant in this study. We found that overexpression of CLB2 encoding B-type cyclin suppressed the temperature-sensitive growth of the puf5Δ mutant. The puf5Δ clb2Δ double mutant displayed a severe growth defect, suggesting that Puf5 positively regulates the expression of a redundant factor with Clb2 in cell cycle progression. We found that expression of CLB1 encoding a redundant B-type cyclin was decreased in the puf5Δ mutant, and that this decrease of the CLB1 expression contributed to the growth defect of the puf5Δ clb2Δ double mutant. Since Puf5 is a negative regulator of the gene expression, we hypothesized that Puf5 negatively regulates the expression of a factor that represses CLB1 expression. We found such a repressor, Ixr1, which is an HMGB (High Mobility Group box B) protein. Deletion of IXR1 restored the decreased expression of CLB1 caused by the puf5Δ mutation and suppressed the growth defect of the puf5Δ clb2Δ double mutant. The expression of IXR1 was negatively regulated by Puf5 in an IXR1 3´ UTR-dependent manner. Our results suggest that IXR1 mRNA is a physiologically important target of Puf5, and that Puf5 and Ixr1 contribute to the cell cycle progression through the regulation of the cell cycle-specific expression of CLB1.     

KIT (c-kit oncogene product) pathway is constitutively activated in human testicular germ cell tumors

We investigated the expression of KIT (product of c-kit oncogene), gain-of-function mutations, and activation of its downstream signal transduction in human testicular cancers. KIT was expressed in 88% (22/25) of seminomas and in 44.4% (4/9) of non-seminomas compared to adjacent normal testicular tissue. Nine of the KIT-expressing seminomas had mutations (40.9%; 9/22) in the c-kit gene; two cases in exon 11 and 7 cases in exon 17. Two of these mutations in exon 17 were novel, and the other seven mutations were identical to the already known gain-of-function mutations which cause activation of KIT without ligand stem cell factor. All of the mutant KIT and 53.8% (7/13) of wild-type KIT were phosphorylated (activated) and associated with phosphorylated phosphatidylinositol 3-kinase (PI3K). Akt was also phosphorylated in these seminomas, suggesting that the KIT-PI3K-Akt pathway is activated in seminoma. These findings suggest that the KIT-PI3K-Akt pathway is constitutively activated in testicular germ cell tumors, due to overexpression of KIT protein and/or gain-of-function mutations in the c-kit gene.     

Structure of a conserved retroviral RNA packaging element by NMR spectroscopy and cryo-electron tomography

The 5′-untranslated regions of all gammaretroviruses contain a conserved “double-hairpin motif” (Ψ^CD) that is required for genome packaging. Both hairpins (SL-C and SL-D) contain GACG tetraloops that, in isolated RNAs, are capable of forming “kissing” interactions stabilized by two intermolecular G-C base pairs. We have determined the three-dimensional structure of the double hairpin from the Moloney murine leukemia virus ([Ψ^CD]₂, 132 nt, 42.8 kDa) using a ²H-edited NMR-spectroscopy-based approach. This approach enabled the detection of ¹H-¹H dipolar interactions that were not observed in previous studies of isolated SL-C and SL-D hairpin RNAs using traditional ¹H-¹H correlated and ¹H-¹³C-edited NMR methods. The hairpins participate in intermolecular cross-kissing interactions (SL-C to SL-D′ and SLC′ to SL-D) and stack in an end-to-end manner (SL-C to SL-D and SL-C′ to SL-D′) that gives rise to an elongated overall shape (ca 95 Å × 45 Å ×  25 Å). The global structure was confirmed by cryo-electron tomography (cryo-ET), making [Ψ^CD]₂ simultaneously the smallest RNA to be structurally characterized to date by cryo-ET and among the largest to be determined by NMR. Our findings suggest that, in addition to promoting dimerization, [Ψ^CD]₂ functions as a scaffold that helps initiate virus assembly by exposing a cluster of conserved UCUG elements for binding to the cognate nucleocapsid domains of assembling viral Gag proteins.     

Effects of one-legged endurance training on femoral arterial and venous size in healthy humans.

The cross-sectional area (CSA) of large-conductance arteries increases in response to endurance training in humans. To determine whether training-induced changes in arterial structure are systemic in nature or, rather, are confined to the arteries supplying exercising muscles, we studied 10 young men who performed one-legged cycle training [80% of one-legged peak O2 uptake (VO2 peak)), 40 min/day, 4 days/wk] for 6 wk and detraining for another 6 wk. There were no significant differences in baseline one-legged VO2 peak) and CSA of the common femoral artery and vein (via B-mode ultrasound) between experimental and control legs. In the experimental leg, one-legged VO2 peak) increased 16% [from 3.0 +/- 0.1 to 3.4 +/- 0.1 (SE) l/min], arterial CSA increased 16% (from 84 +/- 3 to 97 +/- 5 mm2), and venous CSA increased 46% (from 56 +/- 5 to 82 +/- 5 mm2) after endurance training. These changes returned to baseline during detraining. There were no changes in one-legged VO2 peak) and arterial CSA in the control leg, whereas femoral venous CSA in the control leg significantly increased 24% (from 54 +/- 5 to 67 +/- 4 mm2) during training. Changes in femoral arterial and venous CSA in the experimental leg were positively and significantly related to corresponding changes in one-legged VO2 peak) (r = 0.86 and 0.76, respectively), whereas there were no such relations in the control leg (r = 0.10 and 0.17). When stepwise regression analysis was performed, a primary determinant of change in VO2 peak) was change in femoral arterial CSA, explaining approximately 70% of the variability. These results support the hypothesis that the regional increase in blood flow, rather than systemic factors, is associated with the training-induced arterial expansion. Femoral arterial expansion may contribute, at least in part, to improvement in efficiency of blood transport from the heart to exercising muscles and may facilitate achievement of aerobic work capacity.     

Possible involvement of Hcn1 ion channel in learning and memory dysfunction in SAMP8 mice

The senescence-accelerated mouse prone 8 (SAMP8) strain exhibits age-related learning and memory deficits (LMD) at 2 months of age. Combined linkage analysis of 264 F2 intercross SAMP8 × JF1 mice and RNA-seq analysis identified Hcn1 gene out of 29 genes in the LMD region on chromosome 13. Hcn1 in SAMP8 strain showed 15 times less polyglutamine repetition compared to Japanese fancy mouse 1 (JF1). Whole cell patch clamp analysis showed that Hcn1 ion conductivity was significantly lower in SAMP8 compared to that of JF1, which may be associated with learning and memory deficiency.     

Colorimetric dimethyl sulfide sensor using Rhodovulum sulfidophilum cells based on intrinsic pigment conversion by CrtA

A colorimetric whole-cell sensor for dimethyl sulfide (DMS) was constructed based on the in vivo conversion of intrinsic pigments in response to the analyte. In a marine bacterium, Rhodovulum sulfidophilum, carotenoids are synthesized via the spheroidene pathway. In this pathway, demethylspheroidene, a yellow carotenoid, is converted to spheroidene under catalysis of O-methyltransferase. Spheroidene monooxygenase (CrtA) catalyzes the terminal step of the pathway and converts spheroidene to spheroidenone, a red carotenoid. Here, the CrtA gene in R. sulfidophilum was removed and then reintroduced downstream of the DMS dehydrogenase gene promoter. Using this whole-cell sensor, 3 μM DMS or dimethyl sulfoxide can be detected without adding any color-forming reagent. The ratio of the red spheroidenone to total carotenoids increased, as the DMS concentration was raised to 0.3 mM. Comparison of the signal to the background color indicated a shift in the color coordinate from a yellow to a red hue. An intense signal was obtained with 1-day incubation at a high cell density when sensor cells at the exponential growth phase were used. These results show that the genetically engineered R. sulfidophilum cells can be used to monitor the quality of marine aquacultural environments by the naked eye.