Qualitative and quantitative difference in mutation induction between carbon- and neon-ion beams in normal human cells.

We investigated the difference in cell-killing effect and mutation induction between carbon- and neon-ion beams in normal human cells. Carbon- and neon-ion beams were accelerated by the Riken Ring Cyclotron (RRC) at the Institute of Physical and Chemical Research in Japan. Cell-killing effect was measured as the reproductive cell death using the colony formation assay. Mutation induction at the HPRT locus was detected to measure 6-thioguanine-resistant clones. The mutation spectrum of the deletion pattern of exons of induced mutants was analyzed using the multiplex polymerase chain reaction (PCR). Cell-killing effect was almost the same between carbon- and neon-ion beams with similar linear energy transfer (LET) values, while there observed a large difference in mutation frequency. Furthermore, in the case of neon-ion beams 60% of mutants showed total deletions and 35-40% showed partial deletions, while 95-100% of carbon-ion induced mutants showed total deletions. The results suggest that different ion species may cause qualitative and quantitative difference in mutation induction even if the LET values are similar.     

Corelike structures in a stable L-form ofStreptococcus pyogenes

Corelike structures, which were interpreted as straight, large cylinders containing ribosome-like particles surrounded by an amorphous substance of low electron density, were found in a stable L-form ofStreptococcus pyogenes grown in the absence of antibiotics.     

Galnt3 deficiency disrupts acrosome formation and leads to oligoasthenoteratozoospermia

Galnt3 belongs to the GalNAc transferase gene family involved in the initiation of mucin-type O-glycosylation. Male Galnt3-deficient (Galnt3 ^?/?) mice were infertile, as previously reported by Ichikawa et al. (2009). To investigate the involvement of Galnt3 in spermatogenesis, we examined the differentiation of germ cells in Galnt3 ^?/? mice. Galnt3 mRNA was most highly expressed in testis, and Galnt3 protein was localized in the cis-medial parts of the Golgi stacks of spermatocytes and spermatids in the seminiferous tubules. Spermatozoa in Galnt3 ^?/? mice were rare and immotile, and most of them had deformed round heads. They exhibited abnormal acrosome and disturbed mitochondria arrangement in the flagella. At the cap phase, proacrosomal vesicles of various sizes, which had not coalesced to form a single acrosomal vesicle, were attached to the nucleus in Galnt3 ^?/? mice. TUNEL-positive cells were increased in the seminiferous tubules. The binding of VVA lectin, which recognizes the Tn antigen (GalNAc-O-Ser/Thr), in the acrosomal regions of spermatids and spermatozoa in Galnt3 ^?/? mice was drastically reduced. Equatorin is a N, O-sialoglycoprotein localized in the acrosomal membrane and is suggested to be involved in sperm–egg interaction. Immunohistochemical and Western blot analyses showed a drastic reduction in the reactivity with MN9 antibody, which recognizes the O-glycosylated moiety of equatorin and inhibits sperm–egg interaction. These findings indicate that deficiency of Galnt3 results in a severe reduction of mucin-type O-glycans in spermatids and causes impaired acrosome formation, leading to oligoasthenoteratozoospermia, and suggest that Galnt3 may also be involved in the process of fertilization through the O-glycosylation of equatorin.     

The Protective Effects of Levetiracetam on a Human iPSCs-Derived Spinal Muscular Atrophy Model

Neurochemical Research - Spinal muscular atrophy (SMA) is an inherited disease characterized by progressive motor neuron death and subsequent muscle weakness and is caused by deletion or mutation...     

Heavy charged particles produce a bystander effect via cell-cell junctions.

Radiation-induced damage to living cells results from either a direct hit to cellular DNA, or from indirect action which leads to DNA damage from radiation produced radicals. However, in recent years there is evidence that biological effects such as cell killing, mutation induction, chromosomal damage and modification of gene expression can occur in a cell population exposed to low doses of alpha particles. In fact these doses are so low that not all cells in the population will be hit directly by the radiation. Using a precision alpha-particle microbeam, it has been recently demonstrated that irradiated target cells can induce a bystander mutagenic response in neighboring "bystander" cells which were not directly hit by alpha particles. Furthermore, these results suggest that gap-junction mediated cell-to-cell communication plays a critical role in this bystander phenomenon. The purpose of this section is to describe recent studies on bystander biological effects. The recent work described here utilized heavy charged particles for irradiation, and investigated the role of gap-junction mediated cell-cell communication in this phenomenon.     

Evaluation of systemic blood NO dynamics by EPR spectroscopy: HbNO as an endogenous index of NO

The measurement of hemoglobin-nitric oxide (NO) adduct (HbNO) in whole blood by the electron paramagnetic resonance (EPR) method seems relevant for the assessment of systemic NO levels. However, ceruloplasmin and unknown radical species overlap the same magnetic field as that of HbNO. To reveal the EPR spectrum of HbNO, we then introduced the EPR signal subtraction method, which is based on the computer-assisted subtraction of the digitized EPR spectrum of HbNO-depleted blood from that of sample blood using the software. Rats were treated with N(omega)-nitro-L-arginine methyl ester (L-NAME; 120 mg. kg-1. day-1) for 1 wk to obtain HbNO-depleted blood. When this method was applied to the analysis of untreated fresh whole blood, the five-coordinate state of HbNO was observed. HbNO concentration in pentobarbital-anesthetized rats was augmented (change in [HbNO] = 1.6-5.5 microM) by infusion of L-arginine (0.2-0.6 g/kg) but not D-arginine. Using this method, we attempted to evaluate the effects of temocapril on HbNO dynamics in an L-NAME-induced rat endothelial dysfunction model. The oral administration of L-NAME for 2 wk induced a serious hypertension, and the HbNO concentration was reduced (change in [HbNO] = 5.7 microM). Coadministration of temocapril dose dependently improved both changes in blood pressure and the systemic HbNO concentration. In this study, we succeeded in measuring the blood HbNO level as an index of NO by the EPR HbNO signal subtraction method. We also demonstrated that temocapril improves abnormalities of NO dynamics in L-NAME-induced endothelial dysfunction rats using the EPR HbNO signal subtraction method.     

Coumarins and gamma-pyrone derivatives from Prangos pabularia: antibacterial activity and inhibition of cytokine release

The n-hexane and ethyl acetate extracts of the stems and the ethyl acetate extracts of roots from Prangos pabularia afforded an gamma-pyrone derivative and furanocoumarin derivatives with three glucose and gamma-pyrone (pabularin A, B and C), along with 26 previously known compounds (18 coumarins, six terpenoids and two glycosides). Their structures were established on the basis of spectroscopic studies. Of these, 16 coumarin derivatives isolated from P. pabularia were tested for antibacterial activity and inhibition of cytokine release.     

Development of a mouse monoclonal antibody against the chondroitin sulfate-protein linkage region derived from shark cartilage

Glycosaminoglycans (GAGs) like chondroitin sulfate (CS) and heparan sulfate (HS) are synthesized on the tetrasaccharide linkage region, GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser, of proteoglycans. The Xyl can be modified by 2-O-phosphate in both CS and HS, whereas the Gal residues can be sulfated at C-4 and/or C-6 in CS but not in HS. To study the roles of these modifications, monoclonal antibodies were developed against linkage glycopeptides of shark cartilage CS proteoglycans, and one was characterized in detail. This antibody bound hexa- and pentasaccharide-peptides more strongly than unsaturated tetrasaccharide-peptides with the unnatural fourth sugar residue (unsaturated hexuronic acid), suggesting the importance of the fifth and/or fourth saccharide residue GalNAc-5 and/or GlcA-4. Its reactivity was not affected by treatment with chondro-4-sulfatase or alkaline phosphatase, suggesting that 4-O-sulfate on the Gal residues and 2-O-phosphate on the Xyl residue were not recognized. Treatment with weak alkali to cleave the Xyl-Ser linkage completely abolished the binding activity, suggesting the importance of the peptide moiety of the hexasaccharide-peptide for the binding. Based on the amino acid composition and matrix-assisted laser desorption ionization time-of-flight mass spectrometry analyses, it was revealed that the peptide moiety is composed of four amino acids, Ser, Pro, Gly, and Glu. Furthermore, the antibody stained wild-type CHO cells significantly, but much weakly mutant cells deficient in xylosyl- or galactosyltransferase-I required for the biosynthesis of the linkage region. These results suggest that the antibody recognizes the structure GalNAc(±6-O-sulfate)-GlcA-Gal-Gal-Xyl-Ser-(Pro, Gly, Glu). The antibody will be a useful tool for investigating the significance of the linkage region in the biosynthesis and/or intracellular transport of different GAG chains especially since such tools to study the linkage region are lacking.