Immunization of A4galt-deficient mice with glycosphingolipids from renal cell cancers resulted in the generation of anti-sulfoglycolipid monoclonal antibodies

In this study, we immunized Gb3/CD77 synthase gene (A4galt) knockout (KO) mice with glycosphingolipids (GSLs) extracted from 3 renal cell cancer (RCC) cell lines to raise monoclonal antibodies (mAbs) reactive with globo-series GSLs specifically expressed in RCCs. Although a number of mAbs reactive with globo-series GSLs were generated, they reacted with both RCC cell lines and normal kidney cells. When we analyzed recognized antigens by mAbs that were specifically reactive with RCC, but not with normal kidney cells at least on the cell surface, many of them turned out to be reactive with sulfoglycolipids. Eight out of 11 RCC-specific mAbs were reactive with SM2 alone, and the other 3 mAbs were more broadly reactive with sulfated glycolipids, i.e. SM3 and SM4 as well as SM2. In the immunohistochemistry, these anti-sulfoglycolipids mAbs showed RCC-specific reaction, with no or minimal reaction with adjacent normal tissues. Thus, immunization of A4galt KO mice with RCC-derived GSLs resulted in the generation of anti sulfated GSL mAbs, and these mAbs may be applicable for the therapeutics for RCC patients.     

Agatharesinol biosynthesis-related changes of ray parenchyma in sapwood sticks of Cryptomeria japonica during cell death

Planta - The work demonstrates a relationship between the biosynthesis of the secondary metabolite, agatharesinol, and cytological changes that occur in ray parenchyma during cell death in sapwood...     

Axotomy induces axonogenesis in hippocampal neurons by a mechanism dependent on importin β

We characterize the previously unrecognized phenomenon of axotomy-induced axonogenesis in rat embryonic hippocampal neurons in vitro and elucidate the underlying mechanism. New neurites arose from cell bodies after axotomy and grew. These neurites were Tau-1-positive, and the injured axons showed negative immunoreactivity for Tau-1. Axonogenesis was delayed in these neurons by inhibiting the dynein–dynactin complex through the overexpression of p50. Importin β, which was locally translated after axotomy, was associated with the dynein-importin α complex and was required for axonogenesis. Taken together, these results suggest that retrograde transport of injury-induced signals in injured axons play key roles in the axotomy-induced axonogenesis of hippocampal neurons.     

Gait analysis using gravitational acceleration measured by wearable sensors

A novel method for measuring human gait posture using wearable sensor units is proposed. The sensor units consist of a tri-axial acceleration sensor and three gyro sensors aligned on three axes. The acceleration and angular velocity during walking were measured with seven sensor units worn on the abdomen and the lower limb segments (both thighs, shanks and feet). The three-dimensional positions of each joint are calculated from each segment length and joint angle. Joint angle can be estimated mechanically from the gravitational acceleration along the anterior axis of the segment. However, the acceleration data during walking includes three major components; translational acceleration, gravitational acceleration and external noise. Therefore, an optimization analysis was represented to separate only the gravitational acceleration from the acceleration data. Because the cyclic patterns of acceleration data can be found during constant walking, a FFT analysis was applied to obtain some characteristic frequencies in it. A pattern of gravitational acceleration was assumed using some parts of these characteristic frequencies. Every joint position was calculated from the pattern under the condition of physiological motion range of each joint. An optimized pattern of the gravitational acceleration was selected as a solution of an inverse problem. Gaits of three healthy volunteers were measured by walking for 20 s on a flat floor. As a result, the acceleration data of every segment was measured simultaneously. The characteristic three-dimensional walking could be shown by the expression using a stick figure model. In addition, the trajectories of the knee joint in the horizontal plane could be checked by visual imaging on a PC. Therefore, this method provides important quantitive information for gait diagnosis.     

Acetylation of Histone H2AX at Lys 5 by the TIP60 Histone Acetyltransferase Complex Is Essential for the Dynamic Binding of NBS1 to Damaged Chromatin

The association and dissociation of DNA damage response (DDR) factors with damaged chromatin occurs dynamically, which is crucial for the activation of DDR signaling in a spatiotemporal manner. We previously showed that the TIP60 histone acetyltransferase complex acetylates histone H2AX, to facilitate H2AX exchange at sites of DNA damage. However, it remained unclear how the acetylation of histone H2AX by TIP60 is related to the DDR signaling. We found that the acetylation but not the phosphorylation of H2AX is essential for the turnover of NBS1 on damaged chromatin. The loss of H2AX acetylation at Lys 5 by TIP60 in cells disturbed the accumulation of NBS1 at sites of DNA damage. Although the phosphorylation of H2AX is also reportedly required for the retention of NBS1 at damage sites, our data indicated that the acetylation-dependent NBS1 turnover by TIP60 on damaged chromatin restricts the dispersal of NBS1 foci from the sites of DNA damage. These findings indicate the importance of the acetylation-dependent dynamic binding of NBS1 to damaged chromatin, created by histone H2AX exchange, for the proper accumulation of NBS1 at DNA damage sites.     

Mice with defects in HB-EGF ectodomain shedding show severe developmental abnormalities

Heparin-binding EGF-like growth factor (HB-EGF) is first synthesized as a membrane-anchored form (proHB-EGF), and its soluble form (sHB-EGF) is released by ectodomain shedding from proHB-EGF. To examine the significance of proHB-EGF processing in vivo, we generated mutant mice by targeted gene replacement, expressing either an uncleavable form (HBuc) or a transmembrane domain-truncated form (HBdeltatm) of the molecule. HB(uc/uc) mice developed severe heart failure and enlarged heart valves, phenotypes similar to those in proHB-EGF null mice. On the other hand, mice carrying HBdeltatm exhibited severe hyperplasia in both skin and heart. These results indicate that ectodomain shedding of proHB-EGF is essential for HB-EGF function in vivo, and that this process requires strict control.     

Neutralization of toxic heme by Plasmodium falciparum histidine-rich protein 2

Plasmodium falciparum histidine-rich protein 2 (PfHRP2) has been suggested to be an initiator of the polymerization of heme, which is produced as by-product on the digestion of hemoglobin, and a promoter of the H(2)O(2)-induced degradation of heme in food vacuoles of the malarial parasite. In this work, we have designed PfHRP2 model peptides, R18 and R27 (18 and 27 residues, respectively), and used them for optical and electron spin resonance spectroscopic measurements to confirm that the axial ligands of the heme-PfHRP2 complex are the nitrogenous donors derived from the imidazole moieties of histidine residues of PfHRP2. In addition, we revealed that the affinities of R18 and R27 for heme (K(d) = 2.21 x 10(-6) M and 0.71 x 10(-6) M, respectively) might be as high as that of PfHRP2 (K(d) = 0.94 x 10(-6) M). The R27 peptide can remove heme from membrane-intercalated heme and inhibit heme-induced hemolysis. Therefore, we suggest another function of PfHRP2: it may play an important role in the neutralization of toxic heme in the parasite cytoplasm and infected erythrocytes by removing heme from heme-bound membranes or reducing heme-induced hemolysis.     

The Kinetochore Protein Kis1/Eic1/Mis19 Ensures the Integrity of Mitotic Spindles through Maintenance of Kinetochore Factors Mis6/CENP-I and CENP-A

Microtubules play multiple roles in a wide range of cellular phenomena, including cell polarity establishment and chromosome segregation. A number of microtubule regulators have been identified, including microtubule-associated proteins and kinases, and knowledge of these factors has contributed to our molecular understanding of microtubule regulation of each relevant cellular process. The known regulators, however, are insufficient to explain how those processes are linked to one another, underscoring the need to identify additional regulators. To find such novel mechanisms and microtubule regulators, we performed a screen that combined genetics and microscopy for fission yeast mutants defective in microtubule organization. We isolated approximately 900 mutants showing defects in either microtubule organization or the nuclear envelope, and these mutants were classified into 12 categories. We particularly focused on one mutant, kis1, which displayed spindle defects in early mitosis. The kis1 mutant frequently failed to assemble a normal bipolar spindle. The responsible gene encoded a kinetochore protein, Mis19 (also known as Eic1), which localized to the interface of kinetochores and spindle poles. We also found that the inner kinetochore proteins Mis6/CENP-I and Cnp1/CENP-A were delocalized from kinetochores in the kis1 cells and that kinetochore-microtubule attachment was defective. Another mutant, mis6, also displayed similar spindle defects. We conclude that Kis1 is required for inner kinetochore organization, through which Kis1 ensures kinetochore-microtubule attachment and spindle integrity. Thus, we propose an unexpected relationship between inner kinetochore organization and spindle integrity.