Water-soluble gadolinium (Gd) endohedral metallofullerenes have been synthesized as polyhydroxyl forms (Gd@C(82)(OH)(n)(), Gd-fullerenols) and their paramagnetic properties were evaluated by in vivo as well as in vitro for the novel magnetic resonance imaging (MRI) contrast agents for next generation. The in vitro water proton relaxivity, R(1) (the effect on 1/T(1)), of Gd-fullerenols is significantly higher (20-folds) than that of the commercial MRI contrast agent, Magnevist (gadolinium-diethylenetriaminepentaacetic acid, Gd-DTPA) at 1.0 T close to the common field of clinical MRI. This unusually high proton relaxivity of Gd-fullerenols leads to the highest signal enhancement at extremely lower Gd concentration in MRI studies. The strong signal was confirmed in vivo MRI at lung, liver, spleen, and kidney of CDF1 mice after i.v. administration of Gd-fullerenols at a dose of 5 micromol Gd/kg, which was 1/20 of the typical clinical dose (100 micromol Gd/kg) of Gd-DTPA.

Water-soluble multi-hydroxyl lanthanoid (La, Ce, Gd, Dy, and Er) endohedral metallofullerenes (metallofullerenols, M@C(82)(OH)(n)()) have been synthesized and characterized for the use of magnetic resonance imaging (MRI) contrast agents. The observed longitudinal and transverse relaxivities for water protons, r(1) and r(2), of the metallofullerenols are in the range 0.8-73 and 1.2-80 (sec(-1)mM(-1)), respectively, which are significantly higher than those of the corresponding lanthanoid-DTPA chelate complexes. Among these Gd-metallofullerenols, Gd@C(82)(OH)(n)() has exhibited the highest r(1) and r(2) values in consistent with our previous results. The observed large r(1) of the current metallofullerenols can mainly be ascribed to the dipole-dipole relaxation together with a substantial decrease of the overall molecular rotational motion. The large r(2), except for the Gd-metallofullerenols, have been attributed to the so-called Curie spin relaxation. The MRI phantom studies are also performed and are consistent with these results. The metallofullerenols will be an ideal model for future MRI contrast agents with higher proton relaxivities.

To position the mitotic spindle within the cell, dynamic plus ends of astral microtubules are pulled by membrane-associated cortical force-generating machinery. However, in contrast to the chromosome-bound kinetochore structure, how the diffusion-prone cortical machinery is organized to generate large spindle-pulling forces remains poorly understood. Here, we develop a light-induced reconstitution system in human cells. We find that induced cortical targeting of NuMA, but not dynein, is sufficient for spindle pulling. This spindle-pulling activity requires dynein-dynactin recruitment by NuMA’s N-terminal long arm, dynein-based astral microtubule gliding, and NuMA’s direct microtubule-binding activities. Importantly, we demonstrate that cortical NuMA assembles specialized focal structures that cluster multiple force-generating modules to generate cooperative spindle-pulling forces. This clustering activity of NuMA is required for spindle positioning, but not for spindle-pole focusing. We propose that cortical Dynein-Dynactin-NuMA (DDN) clusters act as the core force-generating machinery that organizes a multi-arm ensemble reminiscent of the kinetochore.

Spinal muscular atrophy results from the loss of functional survival motor neuron (SMN1) alleles. Two nearly identical copies of SMN exist and differ only by a single non-polymorphic C to T transition in exon 7. This transition leads to alteration of exon 7 splicing; that is, SMN1 produces a full-length transcript, whereas SMN2 expresses a low level of full-length transcript and predominantly an isoform lacking exon 7. The truncated transcript of SMN encodes a less stable protein with reduced self-oligomerization activity that fails to compensate for the loss of SMN1. In this paper, we identified a cis-acting element (element 1), which is composed of 45 bp in intron 6 responsible for the regulation of SMN exon 7 splicing. Mutations in element 1 or treatment with antisense oligonucleotides directed toward element 1 caused an increase in exon 7 inclusion. An ϳ33-kDa protein was demonstrated to associate with a pre-mRNA sequence containing both element 1 and the C to T transition in SMN exon 7 but not with the sequence containing mutated element 1, suggesting that the binding of the ϳ33-kDa protein plays crucial roles in the skipping of SMN exon 7 containing the C to T transition. Spinal muscular atrophy (SMA)1 is a common autosomal recessive disorder with progressive paralysis caused by the degeneration of motor neurons in the spinal cord (1). The survival of the motor neurons (SMN) gene has been identified as the disease gene of SMA and is present on chromosome 5 at 5q13 (2, 3). Humans contain two nearly identical copies of the SMN gene, SMN1 and SMN2. These genes encode an identical protein, a 294-amino acid RNA-binding protein. Only homozygous deletions or mutations of SMN1 result in the SMA phenotype, and the levels of SMN expression driven by SMN2 in motor neurons inversely correlate with the severity of the disease (4 -15).SMN1 mRNA expresses a full-length transcript, whereas SMN2 produces a low level of full-length transcript and predominantly an isoform lacking exon 7 (SMN⌬7) (2, 16, 17). The SMN⌬7 is less stable (18), and it was reported that SMN⌬7 cannot oligomerize or self-associate as efficiently as the protein produced from the full-length SMN transcript (2,19,20). Therefore, a deficiency in the full-length SMN protein correlates with the disease. The critical difference between SMN1 and SMN2 is a silent nucleotide transition in SMN exon 7. SMN1 contains a C located six nucleotides inside exon 7, whereas SMN2 contains a T at this position. This transition is considered to inhibit one of the splicing regulatory elements within exon 7, which are called exonic splicing enhancers (ESE) (21). A recent report demonstrated the presence of an ESE within exon 7 and that human Tra2-␤1, a member of the serine-arginine-related proteins of splicing factors, binds to the elements and stimulates an ESE (22). However, the critical C to T transition is not contained within the element. Furthermore, the transition does not change the binding activity of Tra2-␤1 to the ESE. Thus, it is still unclear why the C to T trans...

Background : Alterations in homeostasis after various cellular stresses, which prevent protein folding and cause an accumulation of misfolding or malfolding proteins in the endoplasmic reticulum (ER), have the potential to induce cellular damage, and are therefore a type of 'ER stress.' To understand the molecular events or cascades underlying the ER stress response regulated by gene transcription and mediated by stress transducers, it is crucial to identify the molecules induced during ER stress and to analyse the roles of these genes.