The presence of N(ε)-(Carboxymethyl) lysine in the human epidermis

It is well known that advanced glycation end products (AGEs) are formed in long-lived dermal proteins such as collagen, and that their formation is related to skin aging. To examine the distribution of AGEs in skin tissue, we performed immunofluorescence studies on the human skin using an anti-AGEs antibody. Interestingly, AGEs signals were observed not only in the dermis but also in the epidermis. The objectives of this study were to confirm the presence of N(ε)-(Carboxymethyl) lysine (CML), an AGE structure, in the epidermis and to characterize the CML-modified proteins. The presence of CML in the stratum corneum (SC) was examined using liquid chromatography-electrospray ionization time-of-flight mass spectrometry. Concordance between the retention times of a compound in the SC hydrolysate and authentic CML, as well as with the specific mass transition of CML, was detected. This result showed that CML is present in the epidermis. In order to characterize the CML-modified proteins in the epidermis, protein samples extracted from the SC were analyzed using two-dimensional electrophoresis followed by an amino acid sequence analysis. The clarified peptide sequences covered approximately 27% of the amino acid sequences of cytokeratin 10 (K10). In the immunoblotting experiment following the two-dimensional electrophoresis, where protein samples extracted from whole epidermis were used, the position of the major CML-positive spots corresponded to those of K10. Taken together these results showed that CML is present in the human epidermis, and suggest that K10 is one of the target molecules for CML modification in the epidermis.     

Drinking Citrus Fruit Juice Inhibits Vascular Remodeling in Cuff-Induced Vascular Injury Mouse Model

Citrus fruits are thought to have inhibitory effects on oxidative stress, thereby attenuating the onset and progression of cancer and cardiovascular disease; however, there are few reports assessing their effect on vascular remodeling. Here, we investigated the effect of drinking the juice of two different citrus fruits on vascular neointima formation using a cuff-induced vascular injury mouse model. Male C57BL6 mice were divided into five groups as follows: 1) Control (water) (C), 2) 10% Citrus unshiu (CU) juice (CU10), 3) 40% CU juice (CU40), 4) 10% Citrus iyo (CI) juice (CI10), and 5) 40% CI juice (CI40). After drinking them for 2 weeks from 8 weeks of age, cuff injury was induced by polyethylene cuff placement around the femoral artery. Neointima formation was significantly attenuated in CU40, CI10 and CI40 compared with C; however, no remarkable preventive effect was observed in CU10. The increases in levels of various inflammatory markers including cytokines such as monocyte chemotactic protein-1, interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α in response to vascular injury did not differ significantly between C, CU10 and CI10. The increases in cell proliferation and superoxide anion production were markedly attenuated in CI10, but not in CU10 compared with C. The increase in phosphorylated ERK expression was markedly attenuated both in CU10 and CI10 without significant difference between CU10 and CI10. Accumulation of immune cells did not differ between CU10 and CI10. These results indicate that drinking citrus fruit juice attenuates vascular remodeling partly via a reduction of oxidative stress. Interestingly, the preventive efficacy on neointima formation was stronger in CI than in CU at least in part due to more prominent inhibitory effects on oxidative stress by CI.     

A novel costimulation pathway via the 4C8 antigen for the induction of CD4+ regulatory T cells

CD4(+)CD25(+) regulatory T (Treg) cells naturally occur in mice and humans, and similar Treg cells can be induced in vivo and in vitro. However, the molecular mechanisms that mediate the generation of these Treg cell populations remain unknown. We previously described anti-4C8 mAbs that inhibit the postadhesive transendothelial migration of T cells through human endothelial cell monolayers. We demonstrate in this work that Treg cells are induced by costimulation of CD4(+) T cells with anti-CD3 plus anti-4C8. The costimulation induced full activation of CD4(+) T cells with high levels of IL-2 production and cellular expansion that were comparable to those obtained on costimulation by CD28. However, upon restimulation, 4C8-costimulated cells produced high levels of IL-10 but no IL-2 or IL-4, and maintained high expression levels of CD25 and intracellular CD152, as compared to CD28-costimulated cells. The former cells showed hyporesponsiveness to anti-CD3 stimulation and suppressed the activation of bystander T cells depending on cell contact but not IL-10 or TGF-beta. The suppressor cells developed from CD4(+)CD25(-)CD45RO(+) cells. The results suggest that 4C8 costimulation induces the generation of Treg cells that share phenotypic and functional features with CD4(+)CD25(+) T cells, and that CD25(-) memory T cells may differentiate into certain Treg cell subsets in the periphery.     

The simultaneous measurement of nicotinamide adenine dinucleotide and related compounds by liquid chromatography/electrospray ionization tandem mass spectrometry

We have developed a liquid chromatographic-tandem mass spectrometric method that is sensitive and specific and that simultaneously measures cellular NAD(+) and related compounds. Using this method, NAD(+), NAAD, NMN, NAMN, NAM, NA, ADPR, and 5'AMP were first separated over a reverse-phase high-performance liquid chromatography resin in a mobile ammonium formate-methanol linear gradient. Then each compound was ionized at an electrospray source and detected in the positive multiple reaction monitoring mode of a triple-quadrupole tandem mass spectrometer. We found a good linear response for each NAD(+)-related compound. The limits of quantification for NAD(+) and related compounds range from 0.1 to 1 pmol. The extraction efficiency of NAD(+) and related compounds from mouse erythrocytes is between 84 and 114%. The coefficients of variation for the analyses are all less than 6%. Using our method, we measured, in a single analysis, the amounts of NMN, NAMN, NAD(+), and 5'AMP present in mouse erythrocytes. Additionally, this is the first report of a direct determination of the amounts of NMN and NAMN present in any type of cell. These results indicate that our method sensitively, specifically, and simultaneously measures cellular NAD(+) and related compounds.     

Diversity in the Integrase Coding Domain of a gypsy-like Retrotransposon Among Wild Relatives of Rice in the Oryza officinalis Complex

The Oryza officinalis complex is a genetically diverse, tertiary genepool of rice. We analyzed part of the primary structure of the integrase coding domain (ICD) of a gypsy-like retrotransposon from species of the O. officinalis species complex. PCR was performed with degenerate primers that hybridized to conserved sequences in the integrase genes of gypsy-type retrotransposons, using total DNA from different species of the O. officinalis complex as templates. Cloning and sequencing of the PCR products showed that the amplified fragments are highly homologous to each other (75–90%) and belong to one family of retrotransposons that is related to the previously studied RIRE-2 element from rice. Two main subfamilies of 292 and 351 bp were distinguished. Analysis of primary sequence data supports previous reports that sequence divergence during vertical transmission has been the major influence on the evolution of gypsy-type retrotransposons in Oryza species. Based on sequence data phylogenetic relationships among species of the O. officinalis complex were estimated. The data suggests that O. eichingeri is more closely related to the ancestral species of the complex. This revised version was published online in July 2006 with corrections to the Cover Date.     

Expression and physiological role of CCN4/Wnt-induced secreted protein 1 mRNA splicing variants in chondrocytes

CCN4/Wnt-induced secreted protein 1 (WISP1) is one of the CCN (CTGF/Cyr61/Nov) family proteins. CCN members have typical structures composed of four conserved cysteine-rich modules and their variants lacking certain modules, generated by alternative splicing or gene mutations, have been described in various pathological conditions. Several previous reports described a CCN4/WISP1 variant (WISP1v) lacking the second module in a few malignancies, but no information concerning the production of WISP1 variants in normal tissue is currently available. The expression of CCN4/WISP1 mRNA and its variants were analyzed in a human chondrosarcoma-derived chondrocytic cell line, HCS-2/8, and primary rabbit growth cartilage (RGC) chondrocytes. First, we found WISP1v and a novel variant of WISP1 (WISP1vx) to be expressed in HCS-2/8, as well as full-length WISP1 mRNA. This new variant was lacking the coding regions for the second and third modules and a small part of the first module. To monitor the expression of CCN4/WISP1 mRNA along chondrocyte differentiation, RGC cells were cultured and sampled until they were mineralized. As a result, we identified a WISP1v ortholog in normal RGC cells. Interestingly, the WISP1v mRNA level increased dramatically along with terminal differentiation. Furthermore, overexpression of WISP1v provoked expression of an alkaline phosphatase gene that is a marker of terminal differentiation in HCS-2/8 cells. These findings indicate that WISP1v thus plays a critical role in chondrocyte differentiation toward endochondral ossification, whereas HCS-2/8-specific WISP1vx may be associated with the transformed phenotypes of chondrosarcomas.     

Molecular spectrum of somaclonal variation in regenerated rice revealed by whole-genome sequencing

Somaclonal variation is a phenomenon that results in the phenotypic variation of plants regenerated from cell culture. One of the causes of somaclonal variation in rice is the transposition of retrotransposons. However, many aspects of the mechanisms that result in somaclonal variation remain undefined. To detect genome-wide changes in regenerated rice, we analyzed the whole-genome sequences of three plants independently regenerated from cultured cells originating from a single seed stock. Many single-nucleotide polymorphisms (SNPs) and insertions and deletions (indels) were detected in the genomes of the regenerated plants. The transposition of only Tos17 among 43 transposons examined was detected in the regenerated plants. Therefore, the SNPs and indels contribute to the somaclonal variation in regenerated rice in addition to the transposition of Tos17. The observed molecular spectrum was similar to that of the spontaneous mutations in Arabidopsis thaliana. However, the base change ratio was estimated to be 1.74 × 10(-6) base substitutions per site per regeneration, which is 248-fold greater than the spontaneous mutation rate of A. thaliana.     

Unique patterns of pelvic fin evolution: A case study of balistoid fishes (Pisces: Tetraodontiformes) based on whole mitochondrial genome sequences

Balistoid fishes have a unique and reduced pelvic fin structure, which does not exhibit paired structures. The pelvic complex exhibits reductive trends, but its rudimentary structure was retained among balistoids, and its unidirectional and parsimonious reduction in more derived lineages has been hypothesized based on morphology. We investigated the evolution of pelvic complex reduction in balistoids using whole mitochondrial genome (mitogenome) data from 33 species (27 newly determined during the study) that represent the entire morphological diversity of balistoids. Partitioned maximum likelihood and Bayesian analyses were conducted with two datasets that comprised concatenated nucleotide sequences from 13 protein-coding genes (all positions included; third codon positions converted into purine [R] and pyrimidine [Y] [RY-coding]) plus 22 transfer RNA and two ribosomal RNA genes. The resultant trees were well resolved and largely congruent, with most internal branches having high support values. The mitogenomic datasets strongly supported monophylies of both balistids and monacanthids, but rejected previous hypotheses on the intra-relationships in each family. The present tree topology revealed that highly reduced pelvic complexes had multiple origins, and optimization of the traits on the resultant tree strongly suggested the non-unidirectional and independent reduction of pelvic complexes in balistoids. The evolution of balistoid pelvic structure is very different among fishes that exhibit its reductive trends, and this uniqueness in pelvic evolution may be a link to their reproductive behaviors.