Plant nuclear tRNAMet genes are ubiquitously interrupted by introns

We have isolated three independent clones for nuclear elongator tRNA^Met genes from an Arabidopsis DNA library using a tRNA^Met-specific probe generated by PCR. Each of the coding sequences for tRNA^Met in these clones is identical and is interrupted by an identical 11 bp long intervening sequence at the same position in the anticodon loop of the tRNA. Their sequences differ at two positions from the intron in a soybean counterpart. Southern analysis of Arabidopsis DNA demonstrates that a gene family coding for tRNA^Met is dispersed at at least eight loci in the genome. The unspliced precursor tRNA^Met intermediate was detected by RNA analysis using an oligonucleotide probe complementary to the putative intron sequence. In order to know whether introns commonly interrupt plant tRNA^Met genes, their coding sequences were PCR-amplified from the DNAs of eight phylogenetically separate plant species. All 53 sequences determined contain 10 to 13 bp long intervening sequences, always positioned one base downstream from the anticodon. They can all be potentially folded into the secondary structure characteristic for plant intron-containing precursor tRNAs. Surprisingly, GC residues are always present at the 5-distal end of each intron.     

Degradation of Bis(4-Hydroxyphenyl)Methane (Bisphenol F) by Sphingobium yanoikuyae Strain FM-2 Isolated from River Water

Three bacteria capable of utilizing bis(4-hydroxyphenyl)methane (bisphenol F [BPF]) as the sole carbon source were isolated from river water, and they all belonged to the family Sphingomonadaceae. One of the isolates, designated Sphingobium yanoikuyae strain FM-2, at an initial cell density of 0.01 (optical density at 600 nm) completely degraded 0.5 mM BPF within 9 h without any lag period under inductive conditions. Degradation assays of various bisphenols revealed that the BPF-metabolizing system of strain FM-2 was effective only on the limited range of bisphenols consisting of two phenolic rings joined together through a bridging carbon without any methyl substitution on the rings or on the bridging structure. A BPF biodegradation pathway was proposed on the basis of metabolite production patterns and identification of the metabolites. The initial step of BPF biodegradation involves hydroxylation of the bridging carbon to form bis(4-hydroxyphenyl)methanol, followed by oxidation to 4,4′-dihydroxybenzophenone. The 4,4′-dihydroxybenzophenone appears to be further oxidized by the Baeyer-Villiger reaction to 4-hydroxyphenyl 4-hydroxybenzoate, which is then cleaved by oxidation to form 4-hydroxybenzoate and 1,4-hydroquinone. Both of the resultant simple aromatic compounds are mineralized.     

Overexpression of klotho protein modulates uninephrectomy-induced compensatory renal hypertrophy by suppressing IGF-I signals

The cyclin-dependent kinase (CDK) inhibitor p21 plays key roles in p53-dependent DNA-damage responses, i.e., cell cycle checkpoints, senescence, or apoptosis. p21 might also play a role in DNA repair. p21 foci arise at heavy-ion-irradiated DNA-double-strand break (DSB) sites, which are mainly repaired by nonhomologous DNA-end-joining (NHEJ). However, no mechanisms of p21 accumulation at double-strand break (DSB) sites have been clarified in detail. Recent works indicate that Ku70 and Ku80 are essential for the accumulation of other NHEJ core factors, e.g., DNA-PKcs, XRCC4 and XLF, and other DNA damage response factors, e.g., BRCA1. Here, we show that p21 foci arise at laser-irradiated sites in cells from various tissues from various species. The accumulation of EGFP-p21 was detected in not only normal cells, but also transformed or cancer cells. Our results also showed that EGFP-p21 accumulated rapidly at irradiated sites, and colocalized with the DSB marker γ-H2AX and with the DSB sensor protein Ku80. On the other hand, the accumulation occurred in Ku70-, Ku80-, or DNA-PKcs-deficient cell lines and in human papillomavirus 18-positive cells, whereas the p21 mutant without the PCNA-binding region (EGFP-p21(1–146)) failed to accumulate at the irradiated sites. These findings suggest that the accumulation of p21, but not functional p53 and the NHEJ core factors, is dependent on PCNA. These findings also suggest that the accumulation activity of p21 at DNA damaged sites is conserved among human and animal cells, and p21 is a useful tool as a detection marker of DNA damaged sites.     

A centromeric DNA sequence colocalized with a centromere-specific histone H3 in tobacco

Centromeres play an important role in segregating chromosomes into daughter cells, and centromeric DNA assembles specific proteins to form a complex referred to as the kinetochore. Among these proteins, centromere-specific histone H3 (CENH3) is one of the most characterized and found to be located only on active centromeres. We isolated four different CENH3-coding complementary DNAs (cDNAs), two from Nicotiana tabaccum and one each from the ancestral diploid species, Nicotiana sylvestris and Nicotiana tomentosiformis and raised an antibody against N-terminal amino acid sequences deduced from the cDNAs. Immunostaining with the antibody revealed the preferential centromere localization, indicating that the cDNAs cloned in this study encode authentic tobacco CENH3. A tobacco centromeric DNA sequence (Nt2-7) was also identified by chromatin immunoprecipitation cloning using the antibody. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A self-calibrating telemetry system for measurement of ventricular pressure-volume relations in conscious, freely moving rats

Using Bluetooth wireless technology, we developed an implantable telemetry system for measurement of the left ventricular pressure-volume relation in conscious, freely moving rats. The telemetry system consisted of a pressure-conductance catheter (1.8-Fr) connected to a small (14-g) fully implantable signal transmitter. To make the system fully telemetric, calibrations such as blood resistivity and parallel conductance were also conducted telemetrically. To estimate blood resistivity, we used four electrodes arranged 0.2 mm apart on the pressure-conductance catheter. To estimate parallel conductance, we used a dual-frequency method. We examined the accuracy of calibrations, stroke volume (SV) measurements, and the reproducibility of the telemetry. The blood resistivity estimated telemetrically agreed with that measured using an ex vivo cuvette method (y=1.09x - 11.9, r2= 0.88, n=10). Parallel conductance estimated by the dual-frequency (2 and 20 kHz) method correlated well with that measured by a conventional saline injection method (y=1.59x - 1.77, r2= 0.87, n=13). The telemetric SV closely correlated with the flowmetric SV during inferior vena cava occlusions (y=0.96x + 7.5, r2=0.96, n=4). In six conscious rats, differences between the repeated telemetries on different days (3 days apart on average) were reasonably small: 13% for end-diastolic volume, 20% for end-systolic volume, 28% for end-diastolic pressure, and 6% for end-systolic pressure. We conclude that the developed telemetry system enables us to estimate the pressure-volume relation with reasonable accuracy and reproducibility in conscious, untethered rats.     

A novel framework of circulatory equilibrium

A novel framework of circulatory equilibrium was developed by extending Guyton's original concept. In this framework, venous return (CO(V)) for a given stressed volume (V) was characterized by a flat surface as a function of right atrial pressure (P(RA)) and left atrial pressure (P(LA)) as follows: CO(V) = V/W - G(S)P(RA) - G(P)P(LA), where W, G(S), and G(P) denote linear parameters. In seven dogs under total heart bypass, CO(V), P(RA), P(LA), and V were varied to determine the three parameters in each animal with use of multivariate analysis. The coefficient of determination (r(2) = 0.92-0.99) indicated the flatness of the venous return surface. The averaged surface was CO(V) = V/0.129 - 19.61P(RA) - 3.49P(LA). To examine the invariability of the surface parameters among animals, we predicted the circulatory equilibrium in response to changes in stressed volume in another 12 dogs under normal and heart failure conditions. This was achieved by equating the standard surface with the individually measured cardiac output (CO) curve. In this way, we could predict CO [y = 0.90x + 5.6, r(2) = 0.95, standard error of the estimate (SEE) = 8.7 ml.min(-1).kg(-1)], P(RA) (y = 0.96x, r(2) = 0.98, SEE = 0.2 mmHg), and P(LA) (y = 0.89x + 0.5, r(2) = 0.98, SEE = 0.8 mmHg) reasonably well. We conclude that the venous return surface accurately represents the venous return properties of the systemic and pulmonary circulations. The characteristics of the venous return surface are invariable enough among animals, making it possible to predict circulatory equilibrium, even if those characteristics are unknown in individual animals.