Various types of lipid membrane-incorporated C60 with high C60 concentrations can be prepared easily in several hours using the C60 exchange method and the photocleaving activity of cationic lipid membrane-incorporated C60 was appreciably higher than that of the C60.gamma-CDx complex.

Intracellular uptake of a lipid-membrane-incorporated C(60) with a cationic surface into HeLa cells was found to induce cell death under visible light irradiation in high efficiency.

The gap junctional intercellular communication mediated by Cx43 plays indispensable roles in both germ line development and postnatal folliculogenesis. In this study, we focused on the e¡ect of follicle-stimulating hormone (FSH) on the Cx43 protein in rat primary granulosa cells and found that FSH stimulation elevated the phosphorylation in addition to the protein level of Cx43. Serine residues in the carboxyl-terminal region were exclusively phosphorylated in this system and we identi¢ed Ser365, Ser368, Ser369 and Ser373 as major phosphorylation sites by FSH stimulation. A Cx43 variant containing mutations at all these serine residues was found to severely reduce dye transfer activity when assayed in HeLa cells. The present study revealed a novel regulatory mechanism of Cx43-mediated gap junctional intercellular communication through phosphorylation in the carboxyl-terminus.

Abstract. Connexin 43 (Cx43)-mediated gap junctional communication in granulosa cells is crucial for germ line development and postnatal folliculogenesis. We previously showed that folliclestimulating hormone (FSH) promoted phosphorylation of Cx43 in rat primary granulosa cells. We further identified Ser365, Ser368, Ser369, and Ser373 in the carboxy-terminal tail as the major sites of phosphorylation by FSH, and found that the phosphorylation of these residues was essential for channel activity. In this study, we investigated the protein kinase(s) responsible for FSH-induced phosphorylation. H89, a cyclic AMP-dependent protein kinase (PKA) inhibitor, inhibited FSHinduced phosphorylation both in vivo and in vitro, whereas PD98059, a mitogen-activated protein kinase kinase (MEK) inhibitor, had little effect on the phosphorylation level. Ca 2+ -dependent protein kinase (PKC) appeared to negatively regulate phosphorylation. Phosphopeptide mapping with or without H89 treatment indicated that PKA could be responsible for phosphorylation of the four serine residues. In addition, the purified catalytic subunit of PKA could phosphorylate the recombinant Cterminal region of Cx43, but not the variant in which all four serine residues were substituted with alanine. These results suggest that FSH positively regulates Cx43-mediated channel formation and activity through phosphorylation of specific sites by PKA.

Unmodified [60]fullerenes (C60) were solubilised with high stability using various type of poly(ethylene glycol) (PEG) based block copolymer micelles. Block copolymer micelle-incorporated C60 fullerenes were studied in cultures for biological activities using human cervical cancer HeLa cells. As a result, the cationic block copolymer micelles delivered C60 into the cells depending on their surface densities and showed cytotoxicity under photoirradiation.

Although the physiological role of relaxin (RLN) in males remains largely unknown, there is limited evidence that the testis might be a candidate source and target of RLN in boars, as RLN transcripts are detected in the boar testis and it contains RLN-binding sites. To determine whether the boar testis acts as a source and target tissue of RLN, we characterised the expression pattern and cellular localisation of both RLN and its own receptor LGR7 (RXFP1) in boar testes during postnatal development by molecular and immunological approaches. Testes were collected from Duroc boars, and partial cDNA sequences of the boar homologue of human RXFP1 were identified. RLN expression increased through puberty onwards, while RXFP1 expression changed little during development.RLN mRNA and protein expression were restricted to the Leydig cells, whereas both Leydig cells and seminiferous epithelial cells expressed RXFP1 mRNA and protein.Interestingly, RLN was expressed in the testis as an 18 kDa form (the expected size of proRLN), but not as the 6 kDa mature form, during development because of a lack of the enzyme required for proRLN processing. In contrast, RXFP1 was detected at all stages as specific bands of 75 and 91-95 kDa (likely non-glycosylated and glycosylated RXFP1 respectively). Thus, we provide evidence for expression of RLN-RXFP1 ligand-receptor system in the boar testis, suggesting that the testis act as a source and possible target tissue of RLN.

We previously identified solute carrier 22a14 (Slc22a14) as a spermatogenesis-associated transmembrane protein in mice. Although Slc22a14 is a member of the organic anion/cation transporter family, its expression profile and physiological role have not been elucidated. Here, we show that Slc22a14 is crucial for sperm motility and male fertility in mice. Slc22a14 is expressed specifically in male germ cells, and mice lacking the Slc22a14 gene show severe male infertility. Although the overall differentiation of sperm was normal, Slc22a14−/− cauda epididymal spermatozoa showed reduced motility with abnormal flagellar bending. Further, the ability to migrate into the female reproductive tract and fertilise the oocyte were also impaired in Slc22a14−/− spermatozoa. The abnormal flagellar bending was thought to be partly caused by osmotic cell swelling since osmotic challenge or membrane permeabilisation treatment alleviated the tail abnormality. In addition, we found structural abnormalities in Slc22a14−/− sperm cells: the annulus, a ring-like structure at the mid-piece–principal piece junction, was disorganised, and expression and localisation of septin 4, an annulus component protein that is essential for the annulus formation, was also impaired. Taken together, our results demonstrated that Slc22a14 plays a pivotal role in normal flagellar structure, motility and fertility in mouse spermatozoa.

We found that a chemokine receptor gene, CCR1, acts downstream of NFAT2 in RANKLstimulated RAW264 and bone marrow cells. The upstream regulatory region of CCR1 showed RANKLdependent and CsA-suppressible promoter activity. Downregulation of the expression and function of CCR1 suppressed cell migration. Introduction:We previously reported that the expression of NFAT2 induced by RANKL is a key process for progression to multinucleated cells in an in vitro osteoclastogenesis system. Identifying the target genes of NFAT2 would thus be informative about the differentiation process. We focused here on chemokine and chemokine receptor genes that act downstream of NFAT2 in RAW264 cells as well as osteoclast precursors prepared from bone marrow cells. Materials and Methods: RAW264 mouse monocyte/macrophage line cells were cultured with or without cyclosporin A (CsA) in the presence of RANKL or glutathione S-transferase (GST). Osteoclast precursors were prepared from bone marrow cells. RANKL-inducible and CsA-suppressible genes were searched for by microarray analysis, and expression was confirmed by quantitative RT-PCR. Promoter activity was measured by luciferase gene reporter assay. Short interfering (si)RNA for CCR1 was introduced in RAW264 cells. Cell migration activity was examined using a Boyden chamber assay. Results and Conclusions:We identified the chemokine receptor gene CCR1 as a gene showing significant differential expression profiles in osteoclastogenesis in the presence versus the absence of CsA, an inhibitor of NFAT. This property was unique to CCR1 among the chemokine and chemokine receptor genes examined in both RAW264 and bone marrow cells. The upstream regulatory region was isolated from CCR1, and its RANKL-dependent and CsA-suppressible promoter activity was confirmed. The functional significance of CCR1 was assessed by monitoring the migration of cells in a transwell migration assay, and this activity was abolished when either CsA-or CCR1 siRNA-treated cells were used. Moreover, treatment with a G␣ inhibitor pertussis toxin (PTX) or methiolynated-regulated on activation, normal T cells expressed and secreted (Met-RANTES), an antagonist of CCR1, suppressed multinucleated cell formation in the bone marrow cell system. Together, these results suggest that the CCR1 signaling cascade is under the control of NFAT2 and seems to enhance the migration of differentiating osteoclasts.