Dinucleosome DNA of human K562 cells: experimental and computational characterizations

Dinucleosome formation is the first step in the organization of the higher order chromatin structure. With the ultimate aim of elucidating the dinucleosome structure, we constructed a library of human dinucleosome DNA. The library consists of PCR-amplifiable DNA fragments obtained by treatment of nuclei of erythroid K562 cells with micrococcal nuclease followed by extraction of DNA and adaptor ligation to the blunt-ended DNA fragments. The library was then cloned using a plasmid vector and the sequences of the clones were determined. The dominating clones containing the Alu elements were removed. A total of 1002 clones, which comprised a dinucleosome database, contained 84 and 918 clones from the clones before and after removing Alu elements, respectively. Approximately 70% of the clones were between 300 and 400 bp in size and they were distributed to various locations of all chromosomes except the Y chromosome. The clones containing A(2)N(8)A(2)N(8)A(2) or T(2)N(8)T(2)N(8)T(2) sequences were classified into three types, Type I (N shape), Type II (V shape) and Type III (M shape) according to DNA curvature plots. The locations of experimentally determined curved DNA segments matched well with the calculated ones though the clones of Types I and III showed additional curved DNA segments as revealed by the curvature plots. The distributions of complementary dinucleotides in the nucleosome DNA, at the ends of the dinucleosome DNA clones, allowed us to predict the positions of the nucleosome dyad axis, and estimate the size of the nucleosome core DNA, 125nt. The distributions of AA and TT dinucleotides, as well as other RR and YY dinucleotides, showed a periodicity with an average period of 10.4 bases, close to the values observed before. Mapping of nucleosome positions in the dinucleosome database based on the observed periodicity revealed that the nucleosomes were separated by a linker of 7.5+ approximately 10 x n nt. This indicates that the nucleosome-nucleosome orientations are, typically, halfway between parallel and antiparallel. Also an important finding is that the distributions of AA/TT and other RR/YY dinucleotides, apparently, reflect both DNA curvature and DNA bendability, cooperatively contributing to the nucleosome formation.     

Inportance of phosphatidylinositol 3-phosphate in sporulation of Schizosaccharomyces pombe

In Schizosaccharomyces pombe, Pik3p phosphorylates phosphatidylinositol (PI) to produce PI 3-P, which is further phosphorylated by Ste12p to yield PI 3,5-P2. Pik3p is required for both conjugation and sporulation. To test which of PI 3-P and PI 3,5-P2 is required for sporulation, diploid cells defective in production of PI 3,5-P2 were used. They underwent sporulation almost normally provided that the osmotic pressure of the medium was controlled, suggesting that not PI 3,5-P2 but PI 3-P was important. Electron microscopic analysis confirmed normal sporulation in the absence of PI 3,5-P2 although the forespore membrane was found to be less dense in these cells.     

Differing roles of Akt and serum- and glucocorticoid-regulated kinase in glucose metabolism,DNA synthesis,and oncogenic activity

Serum- and glucocorticoid-regulated kinase (SGK) is a serine kinase that has a catalytic domain homologous to that of Akt, but lacks the pleckstrin homology domain present in Akt. Akt reportedly plays a key role in various cellular actions, including glucose transport, glycogen synthesis, DNA synthesis, anti-apoptotic activity, and cell proliferation. In this study, we attempted to reveal the different roles of SGK and Akt by overexpressing active mutants of Akt and SGK. We found that adenovirus-mediated overexpression of myristoylated (myr-) forms of Akt resulted in high glucose transport activity in 3T3-L1 adipocytes, phosphorylated glycogen synthase kinase-3 (GSK3) and enhanced glycogen synthase activity in hepatocytes, and the promotion of DNA synthesis in interleukin-3-dependent 32D cells. In addition, stable transfection of myr-Akt in NIH3T3 cells induced an oncogenic transformation in soft agar assays. The active mutant of SGK (D-SGK, substitution of Ser422 with Asp) and myr-SGK were shown to phosphorylate GSK3 and to enhance glycogen synthase activity in hepatocytes in a manner very similar to that observed for myr-Akt. However, despite the comparable degree of GSK3 phosphorylation between myr-Akt and d-SGK or myr-SGK, d-SGK and myr-SGK failed to enhance glucose transport activity in 3T3-L1 cells, DNA synthesis in 32D cells, and oncogenic transformation in NIH3T3 cells. Therefore, the different roles of SGK and Akt cannot be attributed to ability or inability to translocate to the membrane thorough the pleckstrin homology domain, but rather must be attributable to differences in the relatively narrow substrate specificities of these kinases. In addition, our observations strongly suggest that phosphorylation of GSK3 is either not involved in or not sufficient for GLUT4 translocation, DNA synthesis, or oncogenic transformation. Thus, the identification of substrates selectively phosphorylated by Akt, but by not SGK, may provide clues to clarifying the pathway leading from Akt activation to these cellular activities.     

Activation of AMPK is essential for AICAR-induced glucose uptake by skeletal muscle but not adipocytes

5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) reportedly activates AMP-activated protein kinase (AMPK) and stimulates glucose uptake by skeletal muscle cells. In this study, we investigated the role of AMPK in AICAR-induced glucose uptake by 3T3-L1 adipocytes and rat soleus muscle cells by overexpressing wild-type and dominant negative forms of the AMPKalpha2 subunit by use of adenovirus-mediated gene transfer. Overexpression of the dominant negative mutant had no effect on AICAR-induced glucose transport in adipocytes, although AMPK activation was almost completely abolished. This suggests that AICAR-induced glucose uptake by 3T3-L1 adipocytes is independent of AMPK activation. By contrast, overexpression of the dominant negative AMPKalpha2 mutant in muscle markedly suppressed both AICAR-induced glucose uptake and AMPK activation, although insulin-induced uptake was unaffected. Overexpression of the wild-type AMPKalpha2 subunit significantly increased AMPK activity in muscle but did not enhance glucose uptake. Thus, although AMPK activation may not, by itself, be sufficient to increase glucose transport, it appears essential for AICAR-induced glucose uptake in muscle.     

EMG-angle relationship of the hamstring muscles during maximum knee flexion.

The aim of the present study was to investigate the EMG-joint angle relationship during voluntary contraction with maximum effort and the differences in activity among three hamstring muscles during knee flexion. Ten healthy subjects performed maximum voluntary isometric and isokinetic knee flexion. The isometric tests were performed for 5 s at knee angles of 60 and 90 degrees. The isokinetic test, which consisted of knee flexion from 0 to 120 degrees in the prone position, was performed at an angular velocity of 30 degrees /s (0.523 rad/s). The knee flexion torque was measured using a KIN-COM isokinetic dynamometer. The individual EMG activity of the hamstrings, i.e. the semitendinosus, semimembranosus, long head of the biceps femoris and short head of the biceps femoris muscles, was detected using a bipolar fine wire electrode. With isometric testing, the knee flexion torque at 60 degrees knee flexion was greater than that at 90 degrees. The mean peak isokinetic torque occurred from 15 to 30 degrees knee flexion angle and then the torque decreased as the knee angle increased (p<0.01). The EMG activity of the hamstring muscles varied with the change in knee flexion angle except for the short head of the biceps femoris muscle under isometric condition. With isometric contraction, the integrated EMGs of the semitendinosus and semimembranosus muscles at a knee flexion angle of 60 degrees were significantly lower than that at 90 degrees. During maximum isokinetic contraction, the integrated EMGs of the semitendinosus, semimembranosus and short head of the biceps femoris muscles increased significantly as the knee angle increased from 0 to 105 degrees of knee flexion (p<0.05). On the other hand, the integrated EMG of the long head of the biceps femoris muscle at a knee angle of 60 degrees was significantly greater than that at 90 degrees knee flexion with isometric testing (p<0.01). During maximum isokinetic contraction, the integrated EMG was the greatest at a knee angle between 15 and 30 degrees, and then significantly decreased as the knee angle increased from 30 to 120 degrees (p<0.01). These results demonstrate that the EMG activity of hamstring muscles during maximum isometric and isokinetic knee flexion varies with change in muscle length or joint angle, and that the activity of the long head of the biceps femoris muscle differs considerably from the other three heads of hamstrings.     

Isolation and Contraction of the Stress Fiber

Stress fibers were isolated from cultured human foreskin fibroblasts and bovine endothelial cells, and their contraction was demonstrated in vitro. Cells in culture dishes were first treated with a low-ionic-strength extraction solution and then further extracted using detergents. With gentle washes by pipetting, the nucleus and the apical part of cells were removed. The material on the culture dish was scraped, and the freed material was forced through a hypodermic needle and fractionated by sucrose gradient centrifugation. Isolated, free-floating stress fibers stained brightly with fluorescently labeled phalloidin. When stained with anti-α-actinin or anti-myosin, isolated stress fibers showed banded staining patterns. By electron microscopy, they consisted of bundles of microfilaments, and electron-dense areas were associated with them in a semiperiodic manner. By negative staining, isolated stress fibers often exhibited gentle twisting of microfilament bundles. Focal adhesion–associated proteins were also detected in the isolated stress fiber by both immunocytochemical and biochemical means. In the presence of Mg-ATP, isolated stress fibers shortened, on the average, to 23% of the initial length. The maximum velocity of shortening was several micrometers per second. Polystyrene beads on shortening isolated stress fibers rotated, indicating spiral contraction of stress fibers. Myosin regulatory light chain phosphorylation was detected in contracting stress fibers, and a myosin light chain kinase inhibitor, KT5926, inhibited isolated stress fiber contraction. Our study demonstrates that stress fibers can be isolated with no apparent loss of morphological features and that they are truly contractile organelle.     

Growth and the content of laurinterol and debromolaurinterol in Laurencia okamurae (Ceramiales, Rhodophyta)

The growth of Laurencia okamurae and the content of laurinterol and debromolaurinterol were influenced by various factors. Temperature influenced growth raere te with a maximum at 25°C, regardless of daylength. Maturity depended on temperature rather than daylength; the plants grew without maturing at 15°C, while the plants matured within two weeks at 25°C. The plants were able to grow at salinities of 14–50‰ with maximum growth at 26‰. The salinities growth rash;50‰te reduced with decreasing concentration of nitrate below 1.2 × 10^-3 M, and of total phosphate below 7.5× 10^-8 M. Bromide concentration had no effect on growth, and the plants grew without bromide. Variation in temperature between 15–25°C and daylength produced no obvious change in laurinterol and debromolaurinterol contents. On the other hand, an increase in salinity led to an increase for both. The plants cultured in a completely artificial medium, modified ASP₁₂NTA, showed a marked drop in their content of these metabolites. An increase in concentrations of nitrate, total phosphate or bromide did not restore the content. This revised version was published online in June 2006 with corrections to the Cover Date.