Megalin-mediated endocytosis of cystatin C in proximal tubule cells

Serum levels of cystatin C, an endogenous cysteine proteinase inhibitor, are often used as an indicator of glomerular filtration rate. Although it is known that cystatin C is filtered by glomeruli and metabolized in proximal tubule cells (PTC), the precise molecular mechanism underlying this process is undetermined. Using quartz-crystal microbalance analyses, we demonstrate that cystatin C binds directly to megalin, an endocytic receptor in PTC, in a Ca(+)-dependent manner. We also find that cystatin C is endocytosed specifically via megalin in rat yolk sac epithelium-derived L2 cells which share a variety of characteristics with PTC. Finally, in vivo studies using kidney-specific megalin knockout mice provide evidence that megalin mediates proximal tubular uptake of cystatin C. We conclude that megalin is an endocytic receptor of cystatin C in PTC.     

Diverse selective modes among orthologs/paralogs of the chalcone synthase (Chs) gene family of Arabidopsis thaliana and its relative A. halleri ssp. gemmifera

As a model system, Arabidopsis thaliana and its wild relatives have played an important role in the study of genomics and evolution in plants. In this study, we examined the genetic diversity of the chalcone synthase (Chs) gene, which encodes a key enzyme of the flavonoid pathway and is located on chromosome five, as well as two Chs-like genes on the first and fourth chromosomes of Arabidopsis. The objectives of the study are to determine if natural selection operates differentially on the paralogs of the Chs gene family in A. thaliana and Arabidopsis halleri ssp. gemmifera. The mode of selection was inferred from Tajima's D values from noncoding and coding regions, as well as from the ratio of nonsynonymous to synonymous substitutions. Both McDonald-Kreitman and HKA tests revealed the effects of selection on the allelic distribution, except for the chromosome 1 paralog in ssp. gemmifera. The Chs gene on chromosome 5 was under purifying selection in both species. Significant, negative Tajima's D values at synonymous sites and positive Fay and Wu's H values within coding region, plus reduced genetic variability in introns, indicated effects of background selection in shaping the evolution of this gene region in A. thaliana. The Chs paralog on chromosome 1 was under positive selection in A. thaliana, while interspecific introgression and balancing selection determined the fates of the paralog and resulted in high heterogeneity in ssp. gemmifera. Local adaptation differentiated populations of Japan and China at the locus. In contrast, the other Chs-paralog of chromosome 4 was shaped by purifying selection in A. thaliana, while under positive selection in ssp. gemmifera, as indicated by dn/ds>1. Moreover, these contrasting patterns of selection have likely resulted in functional divergence in Arabidopsis, as indicated by radical amino acid substitutions at the chalcone synthase/stilbene synthase motif of the Chs genes. Unlike previous studies of the evolutionary history of A. thaliana, the high levels of genetic diversity in most gene regions of Chs paralogs and nonsignificant Tajima's D in the intron sequences of the Chs gene family in A. thaliana did not reflect the effects of a recent demographic expansion.     

Molecular analysis of RANKL-independent cell fusion of osteoclast-like cells induced by TNF-alpha, lipopolysaccharide, or peptidoglycan

Focusing on the final step of osteoclastogenesis, we studied cell fusion from tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells into multinuclear cells. TRAP-positive mononuclear cells before generation of multinuclear cells by cell fusion were differentiated from RAW264.7 cells by treatment with receptor activator of nuclear factor kappa B ligand (RANKL), and then the cells were treated with lipopolysaccharide (LPS), followed by culturing for further 12 h. LPS-induced cell fusion even in the absence of RANKL. Similarly, tumor necrosis factor (TNF)-alpha and peptidoglycan (PGN) induced cell fusion, but M-CSF did not. The cell fusion induced by RANKL, TNF-alpha, and LPS was specifically blocked by osteoprotegerin (OPG), anti-TNF-alpha antibody, and polymyxin B, respectively. LPS- and PGN-induced cell fusion was partly inhibited by anti-TNF-alpha antibody but not by OPG. When TRAP-positive mononuclear cells fused to yield multinuclear cells, phosphorylation of Akt, Src, extracellular signal-regulated kinase (ERK), p38MAPK (p38), and c-Jun NH2-terminal kinase (JNK) was observed. The specific chemical inhibitors LY294002 (PI3K), PP2 (Src), U0126 (MAPK-ERK kinase (MEK)/ERK), and SP600125 (JNK) effectively suppressed cell fusion, although SB203580 (p38) did not. mRNA of nuclear factor of activated T-cells c1 (NFATc1) and dendritic cell-specific transmembrane protein (DC-STAMP) during the cell fusion was quantified, however, there was no obvious difference among the TRAP-positive mononuclear cells treated with or without M-CSF, RANKL, TNF-alpha, LPS, or PGN. Collectively, RANKL, TNF-alpha, LPS, and PGN induced cell fusion of osteoclasts through their own receptors. Subsequent activation of signaling pathways involving PI3K, Src, ERK, and JNK molecules was required for the cell fusion. Although DC-STAMP is considered to be a requisite for cell fusion of osteoclasts, cell fusion-inducing factors other than DC-STAMP might be necessary for the cell fusion.     

KONJAC1 and 2 Are Key Factors for GDP-Mannose Generation and Affect l-Ascorbic Acid and Glucomannan Biosynthesis in Arabidopsis

Humans are unable to synthesize l-ascorbic acid (AsA), yet it is required as a cofactor in many critical biochemical reactions. The majority of human dietary AsA is obtained from plants. In Arabidopsis thaliana, a GDP-mannose pyrophosphorylase (GMPP), VITAMIN C DEFECTIVE1 (VTC1), catalyzes a rate-limiting step in AsA synthesis: the formation of GDP-Man. In this study, we identified two nucleotide sugar pyrophosphorylase-like proteins, KONJAC1 (KJC1) and KJC2, which stimulate the activity of VTC1. The kjc1kjc2 double mutant exhibited severe dwarfism, indicating that KJC proteins are important for growth and development. The kjc1 mutation reduced GMPP activity to 10% of wild-type levels, leading to a 60% reduction in AsA levels. On the contrary, overexpression of KJC1 significantly increased GMPP activity. The kjc1 and kjc1kjc2 mutants also exhibited significantly reduced levels of glucomannan, which is also synthesized from GDP-Man. Recombinant KJC1 and KJC2 enhanced the GMPP activity of recombinant VTC1 in vitro, while KJCs did not show GMPP activity. Yeast two-hybrid assays suggested that the stimulation of GMPP activity occurs via interaction of KJCs with VTC1. These results suggest that KJCs are key factors for the generation of GDP-Man and affect AsA level and glucomannan accumulation through the stimulation of VTC1 GMPP activity.     

Tumor-selective,adenoviral-mediated GFP genetic labeling of human cancer in the live mouse reports future recurrence after resection

We have previously developed a telomerase-specific replicating adenovirus expressing GFP (OBP-401), which can selectively label tumors in vivo with GFP. Intraperitoneal (i.p.) injection of OBP-401 specifically labeled peritoneal tumors with GFP, enabling fluorescence visualization of the disseminated disease and real-time fluorescence surgical navigation. However, the technical problems with removing all cancer cells still remain, even with fluorescence-guided surgery. In this study, we report imaging of tumor recurrence after fluorescence-guided surgery of tumors labeled in vivo with the telomerase-dependent, GFP-containing adenovirus OBP-401.. Recurrent tumor nodules brightly expressed GFP, indicating that initial OBP-401-GFP labeling of peritoneal disease was genetically stable, such that proliferating residual cancer cells still express GFP. In situ tumor labeling with a genetic reporter has important advantages over antibody and other non-genetic labeling of tumors, since residual disease remains labeled during recurrence and can be further resected under fluorescence guidance.Key words: green fluorescent protein, adenovirus, cancer labeling, in situ, fluorescence-guided surgery, recurrence, detection     

Glycomics of a novel type-2 N-acetyllactosamine-specific lectin purified from the feather star, Oxycomanthus japonicus (Pelmatozoa: Crinoidea)

A lectin - designated OXYL for the purposes of this study that strongly recognizes complex-type oligosaccharides of serum glycoproteins - was purified from a crinoid, the feather star Oxycomanthus japonicus, the most basal group among extant echinoderms. OXYL was purified through a combination of anion-exchange and affinity chromatography using Q-sepharose and fetuin-sepharose gel, respectively. Lectin was determined to be a 14-kDa polypeptide by sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing conditions. However, 14-kDa and 28-kDa bands appeared in the same proportion under non-reducing conditions. Gel permeation chromatography showed a 54-kDa peak, suggesting that lectin consists of four 14-kDa subunits. Divalent cations were not indicated, and stable haemagglutination activity was demonstrated at pH 4-12 and temperatures below 60°C. Surface plasmon resonance analysis of OXYL against fetuin showed k(ass) and k(diss) values of 1.4×10(-6)M(-1)s(-1) and 3.1×10(-3)s(-1), respectively, indicating that it has a strong binding affinity to the glycoprotein as lectin. Frontal affinity chromatography using 25 types of prydylamine-conjugated glycans indicated that OXYL specifically recognizes multi-antennary complex-type oligosaccharides containing type-2 N-acetyllactosamines (Galβ1-4GlcNAc) if α2-3-linked sialic acid is linked at the non-reducing terminal. However, type-1 N-acetyllactosamine (Galβ1-3GlcNAc) chains and α2-6-linked sialic acids were never recognized by OXYL. This profiling study showed that OXYL essentially recognizes β1-4-linkage at C-1 position and free OH group at C-6 position of Gal in addition to the conservation of N-acetyl groups at C-2 position and free OH groups at C-3 position of GlcNAc in N-acetyllactosamine. This is the first report on glycomics on a lectin purified from an echinoderm belonging to the subphylum Pelmatozoa.     

Effects of balance training using wobble boards in the elderly

Few studies have examined balance training of elderly people using wobble boards. This study assessed the effects of wobble board balance training on physical function in institutionalized elderly people. This study examined 23 subjects (age 84.2 ± 5.9 years) who lived in a nursing home. The exercise program for the training group comprised balance training standing on a wobble board for 9 weeks, twice a week. In all, 11 training group subjects and 11 control group subjects completed this study. After 9 weeks, standing time on a wobble board, standing time on a balance mat, and maximum displacement distance of anterior-posterior center of pressure in the training group were significantly greater than those of the control group. Frequency analysis revealed that the power spectrum in 0.1-0.2 Hz significantly increased in the training group. These results suggest that wobble board training is effective for elderly people to improve their standing balance, by which they frequently control their center of gravity and maintain a standing posture on unstable surface conditions.     

Walking strategies during the transition between level and hill surfaces

Healthy young adults transition between level and hill surfaces of various angles while walking at fluctuating speeds. These surface transitions have the potential to decrease dynamic balance in both the anterior-posterior and medial-lateral directions. Hence, the purpose of the current study was to analyze modifications in temporal-spatial parameters during hill walking transitions. We hypothesized that in comparison with level walking, the transition strides would indicate the adoption of a distinct gait strategy with a greater base of support. Thirty-four participants completed level and hill trials on a walkway with a 15-degree portable ramp apparatus. We collected data during 4 transition strides between level and ramp surfaces. In support of our hypothesis, compared with level walking, the base of support was 20% greater during 3 out of the 4 transition strides. In short, our results illustrate that healthy young adults did adopt a distinct gait strategy different from both level and hill walking during transitions strides.     

High levels of human recombinant cyclooxygenase-1 expression in mammalian cells using a novel gene amplification method

We report the expression of a high level of human cyclooxygenase-1 (hCOX-1) in mammalian cells using a novel gene amplification method known as the IR/MAR gene amplification system. IR/MAR-plasmids contain a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) and amplify autonomously without a specific induction process. In this study, the IR/MAR-plasmid pΔBN.AR1 was cotransfected with pCAG-COX1, which expresses hCOX-1, into human HEK293T cells, and G418 and blasticidin S double-resistant cells were obtained in about 1month. Real-time PCR and Western blotting revealed that the expressions of hCOX-1 mRNA and protein in both polyclonal and monoclonal cells were remarkably higher than those in only pCAG-COX1-transfected control cells. Southern blotting demonstrated the amplification of the hCOX-1 gene, and the copy number of clone #43 obtained by the cotransfection of pΔBN.AR1 and pCAG-COX1 was more than 20 copies per cell, though that of clone #14 obtained without using the IR/MAR plasmid pΔBN.AR1 was only two copies. These results indicate that a high level of hCOX-1 expression was achieved as a result of hCOX-1 gene amplification. Furthermore, the crude extract from clone #43 showed a strong COX-1 activity, and the activity was inhibited by the representative COX-1 inhibitor indomethacin, with an IC(50) value of 36nM. These results demonstrate that the IR/MAR gene amplification system is a simple but useful method for generating highly productive mammalian cells.