Dissecting in vivo steady-state dynamics of karyopherin-dependent nuclear transport

Karyopherin-dependent molecular transport through the nuclear pore complex is maintained by constant recycling pathways of karyopherins coupled with the Ran-dependent cargo catch-and-release mechanism. Although many studies have revealed the bidirectional dynamics of karyopherins, the entire kinetics of the steady-state dynamics of karyopherin and cargo is still not fully understood. In this study, we used fluorescence recovery after photobleaching and fluorescence loss in photobleaching on live cells to provide convincing in vivo proof that karyopherin-mediated nucleocytoplasmic transport of cargoes is bidirectional. Continuous photobleaching of the cytoplasm of live cells expressing NLS cargoes led to progressive decrease of nuclear fluorescence signals. In addition, experimentally obtained kinetic parameters of karyopherin complexes were used to establish a kinetic model to explain the entire cargo import and export transport cycles facilitated by importin β. The results strongly indicate that constant shuttling of karyopherins, either free or bound to cargo, ensures proper balancing of nucleocytoplasmic distribution of cargoes and establishes effective regulation of cargo dynamics by RanGTP.     

Development of glutathione-coupled cantilever for the single-molecule force measurement by scanning force microscopy

The accuracy and the fidelity of a single-molecule force measurement largely rely on how the molecule of interest is attached to the solid substrate surface (bead, cantilever, cover glass and etc.). A site-specific attachment of a protein without affecting its structure and enzymatic function has been a major concern. Here, we established a glutathione-coupled cantilever to which any glutathione S-transferase (GST)-fused proteins can be attached in a desired direction. The rupture force between glutathione and GST was approximately 100 pN on average. By using this cantilever, we succeeded in measuring the interaction force between importin alpha and importin beta.     

Fast microscopical dissection of action scenes played by Escherichia coli RNA polymerase

Using fast-scanning atomic force microscopy, we directly visualized the interaction of Escherichia coli RNA polymerase (RNAP) with DNA at the scan rate of 1-2 frames per second. The analyses showed that the RNAP can locate the promoter region not only by sliding but also by hopping and/or segmental transfer. Upon the addition of 0.05 mM NTPs to the stalled complex, the RNAP molecule pulled the template DNA uni-directionally at the rates of 15 nucleotides/s on average. The present method is potentially applicable to examine a variety of protein-nucleic acid interactions, especially those involved in the process of gene regulation.     

Does famine influence sex ratio at birth? Evidence from the 1959–1961 Great Leap Forward Famine in China

The current study examined the long-term trend in sex ratio at birth between 1929 and 1982 using retrospective birth histories of 310 101 Chinese women collected in a large, nationally representative sample survey in 1982. The study identified an abrupt decline in sex ratio at birth between April 1960, over a year after the Great Leap Forward Famine began, and October 1963, approximately 2 years after the famine ended, followed by a compensatory rise between October 1963 and July 1965. These findings support the adaptive sex ratio adjustment hypothesis that mothers in good condition are more likely to give birth to sons, whereas mothers in poor condition are more likely to give birth to daughters. In addition, these findings help explain the lack of consistent evidence reported by earlier studies based on the 1944-1945 Dutch Hunger Winter or the 1942 Leningrad Siege.     

Dynamic structures of Bacillus subtilis RecN-DNA complexes

Genetic and cytological evidences suggest that Bacillus subtilis RecN acts prior to and after end-processing of DNA double-strand ends via homologous recombination, appears to participate in the assembly of a DNA repair centre and interacts with incoming single-stranded (ss) DNA during natural transformation. We have determined the architecture of RecN–ssDNA complexes by atomic force microscopy (AFM). ATP induces changes in the architecture of the RecN–ssDNA complexes and stimulates inter-complex assembly, thereby increasing the local concentration of DNA ends. The large CII and CIII complexes formed are insensitive to SsbA (counterpart of Escherichia coli SSB or eukaryotic RPA protein) addition, but RecA induces dislodging of RecN from the overhangs of duplex DNA molecules. Reciprocally, in the presence of RecN, RecA does not form large RecA–DNA networks. Based on these results, we hypothesize that in the presence of ATP, RecN tethers the 3′-ssDNA ends, and facilitates the access of RecA to the high local concentration of DNA ends. Then, the resulting RecA nucleoprotein filaments, on different ssDNA segments, might promote the simultaneous genome-wide homology search.     

Impaired glucose tolerance without hypertriglyceridemia does not enhance postprandial lipemia.

Postprandial lipemia has been thought to be one of risk factors for coronary heart disease, and enhances in potential patients for atherosclerotic disease. Patients with impaired glucose tolerance (IGT) often show hypertriglyceride, which is caused by enhanced portprandial lipemia. Therefore, postprandial lipemia in patients with IGT and without hypertriglyceridemia has not been cleared. We have examined the levels of plasma triglyceride and chylomicron remnants after a high fat meal load (1250 kcal, 40% fat and 420 mg cholesterol) in 13 normotriglyceridemic subjects with IGT and 10 controls with normal glucose tolerance (NGT). Chylomicron remnants were evaluated as remnant-like particles (RLP) that were not bound to an immunoaffinity gel mixture containing apo A-I and apo B-100 monoclonal antibody. RLP cholesterol levels 4 hours after the fat load were significantly lower in IGT subjects than in NGT subjects. Increase of RLP cholesterol after the fat meal load only significantly correlated with increase of insulin during the first 30 min after a 75 g oral glucose tolerance test, but not fasting lipid, insulinogenic index and HOMA-R (homeostasis model) in all subjects. These results suggest that postprandial response does not enhance in IGT subjects, and may associate with early-phase insulin secretion and without insulin resistance in normotriglyceridemic men with IGT or NGT.     

Linker histone H1 per se can induce three-dimensional folding of chromatin fiber

Higher-order architectures of chromosomes play important roles in the regulation of genome functions. To understand the molecular mechanism of genome packing, an in vitro chromatin reconstitution method and a single-molecule imaging technique (atomic force microscopy) were combined. In 50 mM NaCl, well-stretched beads-on-a-string chromatin fiber was observed. However, in 100 mM NaCl, salt-induced interaction between nucleosomes caused partial aggregation. Addition of histone H1 promoted a further folding of the fiber into thicker fibers 20-30 nm in width. Micrococcal nuclease digestion of these thicker fibers produced an approximately 170 bp fragment of nucleosomal DNA, which was approximately 20 bp longer than in the absence of histone H1 ( approximately 150 bp), indicating that H1 is correctly placed at the linker region. The width of the fiber depended on the ionic strength. Widths of 20 nm in 50 mM NaCl became 30 nm as the ionic strength was changed to 100 mM. On the basis of these results, a flexible model of chromatin fiber formation was proposed, where the mode of the fiber compaction changes depending both on salt environment and linker histone H1. The biological significance of this property of the chromatin architecture will be apparent in the closed segments ( approximately 100 kb) between SAR/MAR regions.