Low-level laser selectively inhibiting colorectal cancer cell metabolic activity and inducing apoptosis for delaying the development of intestinal cancer.

Low-level laser irradiation (LLLI) is a novel approach that shows promise for the treatment of colorectal cancer (CRC). However, the molecular mechanisms underlying its biochemical effects and gene expression remain unclear. Here, LLLI (632.8 nm) was used to treat CRC RKO cells and normal small intestinal NCM460 cells. LLLI showed a significant dose- and time-dependent effect on cell viability, in which a single dose of irradiation at 15 J/cm2 selectively inhibited the growth of RKO cells but largely unaffected the activity of NCM460 cells. And then, LLLI produced an internal response, effectively reducing the level of H2O2 in tumor cells, downregulating the mitochondrial membrane potential, and improving the efficiency of apoptosis in CRC, but no internal response was observed in NCM460 cells under the same conditions. Furthermore, the expression of several important genes in the classical WNT pathway was significantly downregulated, and the pathway was inactivated after LLLI intervention, thereby inhibiting tumor cell growth. Simultaneously, TNF-α was effectively activated to stimulate the caspase family members of the death effector to initiate apoptosis led by the extrinsic pathway. LLLI successfully achieves tumor cell normalization while delivering a potent anticancer effect, expected to be a novel therapeutic modality for CRC.

The involvement of cytokine-like 1 (Cytl1) in chondrogenesis and cartilage metabolism.

The Cytokine-like 1 (Cytl1) is first identified in bone marrow cells and preferentially expressed in cartilaginous tissue, and showed chondrogenic effects in mesenchymal cells, not essential for cartilage or bone development as in Cytl1 knock-out mice but associated with cartilage inflammatory and destruction. Here, we show the involvement of Cytl1 in chondrogenesis. Using specified chondrogenic embryonic skeleton and adult cartilage, the Cytl1 gene expression was investigated with associated chondrogenic factors by quantitative RT-PCR. The effect of Cytl1 protein (rCytl1) on cultured chondrocytes to regulate expression of key factors and phenotypic markers was studied. The results revealed that Cytl1 was highly expressed in chondrogenic process in embryos and adult cartilage. The rCytl1 increased the expression of Sox9 and Col2α1 with stabilized Col1α1 in cultured chondrocytes (redifferentiation). The Cytl1 was expressed and involved at all stages of cartilage development. Furthermore, Cytl1 expression shared similar patterns as other chondrogenic factors, implying interactions with other factors in chondrogenic process. Cytl1 is involved in cartilage development and matrix homeostasis, which defines the dedifferentiation phenotype of chondrocytes, essential to forming of functional cartilage in both physiologic remodeling and pathologic regeneration.

Promoted Viability and Differentiated Phenotype of Cultured Chondrocytes With Low Level Laser Irradiation Potentiate Efficacious Cells for Therapeutics.

Effective clinical treatments of cartilage lesions in affected joints require large numbers of viable chondrogenic cells generated through in vivo stimulation or ex vivo expansion of chondrocytes isolated from small biopsy specimens. Conventional passaging of chondrocytes in culture provides sufficient cells for treatments but these cells usually lose their differentiated phenotype. This leads to the formation of fibrocartilaginous tissue due to a malfunctioning repair process. Biostimulation of passaging chondrocytes with low level laser irradiation (LLLI) may theoretically produce more functional chondrocytes for cell-based repair of cartilage defects. Molecular and cellular analyses, cytochemistry, cell cultivation, and microscopy showed that LLLI treatments were found to (1) increase chondrocyte viability, (2) promote secretion of matrix proteins, (3) upregulate expression of chondrogenic genes, and (4) downregulate gene expression of cell destructive proteases and genes coding for mediators involved in the extrinsic apoptosis signaling pathway. Furthermore, LLLI attenuated induction of genes associated with cell death and matrix breakdown induced by IL-1ß, some of which was seen at the protein level, with verification of effects on gene expression in the C28/I2 human chondrocyte line. LLLI treatments during culture generated larger numbers of viable chondrocytes compared to untreated cultures. Moreover, LLLI-treated chondrocytes in culture also rectified and simultaneously maintained their differentiated phenotype. Cultured chondrocytes treated with LLLI are a promising cell source for repairing cartilage lesions in vivo and restoration of articular function using tissue engineering strategies.

Catalase Enhances Viability of Human Chondrocytes in Culture by Reducing Reactive Oxygen Species and Counteracting Tumor Necrosis Factor-α-Induced Apoptosis.

BACKGROUND/AIMS: Both physiologic remodeling and pathologic regeneration of cartilage tissue rely upon chondrocyte functions and are benefited from factors that promote viability and inhibit apoptosis of the cell, and associated mechanisms. High level of reactive oxygen species (ROS) and proinflammatory cytokines activate apoptosis signaling and initiate cell death, which can be attenuated by antioxidants. This study examined the effect of catalase (CAT) on ROS and tumor necrosis factor-α (TNF-α)-induced apoptosis in human C28/I2 chondrocytes cultured in monolayer. METHODS: Chondrocytes were treated with diluted CAT in the presence or absence of TNF-α and compared to untreated cells. Levels of hydrogen peroxide (H2O2) and mitochondrial membrane potential (Δψm) were measured using fluorescent labeling, cell apoptosis was assayed by flow cytometry using Annexin V/propidium iodide (PI) staining, gene expression was detected by quantitative real time polymerase chain reaction (qRT-PCR) and the proteins were investigated by Western blotting. RESULTS: CAT effectively reduced the intracellular ROS caused by the monolayer culture system, enhanced the Δψm depending on the presence of TNF-α and promoted morphological features at sub-cellular level. CAT also attenuated the TNF-α-upregulated expression of factors/mediators of extrinsic cell death cascade and apoptotic caspases, ultimately resulted in promoted cellular viability. CONCLUSION: The anti-apoptotic effect of CAT on chondrocytes via scavenging ROS and suppressing TNF-α-induced cell apoptosis by TNF/TNF receptor (TNFR) mediated death signaling pathway and potentiate CAT as a complementary agent beneficial to cartilage remodeling and regeneration in vivo, and cell-based therapies of cartilage repair demanding viable cells expanded ex vivo.

Ci-AMBP: a highly conserved member of the microglobulin superfamily of proteinase inhibitors in grass carp, Ctenopharyngodon idellus.

A full-length cDNA clone encoding grass carp (Ctenopharyngodon idellus) α1-microglobulin/bikunin precursor (Ci-AMBP) was isolated by subtracted differential hybridization screening from a liver cDNA library. The deduced amino acid sequence shared approximately 50% sequence identity with its mammalian counterparts, but more than 90% identity with another fish species. AMBPs are the precursors of the plasma glycoproteins α1-microglobulin (α1m) and bikunin. Both peptide structures and their chromosomal organization were well conserved in Ci-AMBP. The α1m and bikunin polypeptides are separated by the typical tetrapeptide R-A-R-R that provides an endoproteolytic cleavage site for maturation. The genetic organization of domains and functional motifs indicated that Ci-AMBP is a typical member of the lipocalin and Kunitz-type protease inhibitor superfamilies. Expression of the Ci-AMBP gene in different tissues/organs was evaluated using semi-quantitative RT-PCR and, in contrast to the restricted expression in other species, transcripts were detected in a wide range of tissues. The most abundant expression occurred in the secretory organs, which supports the roles of α1m and bikunin in the immune response to diseases and in the stress response.

Salvianolic acid B regulates gene expression and promotes cell viability in chondrocytes.

Articular chondrocytes reside in lacunae distributed in cartilage responsible for the remodelling of the tissue with limited ability of damage repairing. The in vitro expanded chondrocytes enhanced by factors/agents to obtain large numbers of cells with strengthened phenotype are essential for successful repair of cartilage lesions by clinical cell implantation therapies. Because the salvianolic acid B (Sal B), a major hydrophilic therapeutic agent isolated from Salvia miltiorrhiza, has been widely used to treat diseases and able to stimulate activity of cells, this study examines the effects of Sal B on passaged chondrocytes. Chondrocytes were treated with various concentrations of Sal B in monolayer culture, their morphological properties and changes, and mitochondrial membrane potential were analysed using microscopic analyses, including cellular biochemical staining and confocal laser scanning microscopy. The proteins were quantified by BCA and Western blotting, and the transcription of genes was detected by qRT-PCR. The passaged chondrocytes treated with Sal B showed strengthened cellular synthesis and stabilized mitochondrial membrane potential with upregulated expression of the marker genes for chondrocyte phenotype, Col2-α1, Acan and Sox9, the key Wnt signalling molecule ß-catenin and paracrine cytokine Cytl-1. The treatments using CYTL-1 protein significantly increased expression of Col2-α1 and Acan with no effect on Sox9, indicating the paracrine cytokine acts on chondrocytes independent of SOX9. Sal B has ultimately promoted cell growth and enhanced chondrocyte phenotype. The chondrocytes treated with pharmaceutical agent and cytokine in the formulated medium for generating large number of differentiated chondrocytes would facilitate the cell-based therapies for cartilage repair.

Repair of massively defected hemi-joints using demineralized osteoarticular allografts with protected cartilage.

Surgical replacement of massively defected joints necessarily relies on osteochondral grafts effective to both of bone and cartilage. Demineralized bone matrix (DBM) retains the osteoconductivity but destroys viable chondrocytes in the cartilage portion essential for successful restoration of defected joints. This study prepared osteochondral grafts of DBM with protected cartilage. Protected cartilage portions was characterized by cellular and molecular biology and the grafts were allogenically used for grafting. Protected cartilage showed similar histomorphological structure and protected proteins estimated by total proteins and cartilage specific proteins as in those of fresh controls when DBMs were generated in bone portions. Such grafts were successfully used for simultaneously repair of bone and cartilage in massively defected osteoarticular joints within 16 weeks post-surgery. These results present an allograft with clinical potential for simultaneous restoration of bone and cartilage in defected joints.

New bone formation and microstructure assessed by combination of confocal laser scanning microscopy and differential interference contrast microscopy.

Bone is a mineralized connective tissue that is continuously and microstructurally remodeled. Altered bone formation and microstructure arise in pathological bone conditions such as osteoporosis, osteonecrosis, fracture repair, and Paget disease of bone. A proper and objective assessment of bone formation and microstructure will provide insight into the understanding of bone pathogenesis and remodeling. Here, new bone formation ex vitro and its microstructure were evaluated in in vivo multiple sequential polychrome-labeled samples using confocal laser scanning microscopy (CLSM), which generated clearer and more reliable images of thick bone sections than conventional fluorescence microscopy (CFM). Intriguingly, fine details of the bone microstructural features, including the mineralization fronts, quiescent versus active osteons, and Volkmann's channel, were elucidated using CLSM, which defines the relationship between morphological changes and function, when combined with differential interference contrast microscopy. Furthermore, CLSM provided objective evaluations of bone formation, such as the ratio of labeled areas of new bone formation in a rabbit model when compared with CFM. Altogether, new bone formation and its microstructure can be evaluated more adequately using a combination of CLSM and DIC microscopies.

Engineering the male-specificity of Fab against SDM antigen by chain shuffling.

High-titer serologically detected male (SDM) antibody fragments are essential for specific binding to the SDM antigen and promoting its application. The A8 clone previously obtained from an original phage antibody library was further affinity-matured by light- and high-chain shuffling respectively, to generate the end product B9 clone. The binding capacity of B9 phage Fabs to male splenocytes doubled the value of its parental A8 clone (determined using ELISA). Based on immunofluorescent staining, B9-Fabs mainly bound to the surface antigen of male splenocytes and recognized testicular cells. The resulting B9-Fabs detected a single protein (approximately 40 kDa determined using Western blot analysis of male splenocytes and testis); its high SDM antigen binding ability might have been because of mutation sites and varied lengths of the amino acid sequences in the complementarity determining regions-3 of the κ and Fd chains. The new recombinant clones of Fab that were phage-enhanced using chain shuffling were candidate molecules for investigating molecular mechanisms of SDM antigens specific binding and applications.

Evolution of the shell coat and yolk in amniotes: a marsupial perspective.

Two characters distinguish oogenesis and early development in marsupials and monotremes: (1) the shell coat that persists from the zygote to somite stages in marsupials or until hatching in monotremes; and (2) the numerous, apparently almost empty vesicles that appear in primary oocytes, increase during oogenesis in marsupials and monotremes before being shed into the cleavage cavity and are preferentially distributed to the trophoblast lineage in marsupials, but comprise the latebra in monotremes. Analysis of these unusual characters used Southern analysis of genomic DNA dot blots and histology and electron microscopy. The evidence suggests that the marsupial shell coat protein, CP4, was probably characteristic of the egg of the mammalian ancestor. Further, the vesicles, present in marsupials during oogensis and cleavage and in eutherian mammals during blastocyst formation are the residual elements of white yolk present in the larger yolky eggs of monotemes and sauropsids. By comparison with the function of the vesicle components in marsupials, it is suggested that one role for the white yolk in monotremes and the sauropsids is to provide extracellular matrix (ECM), especially hyaluronan containing stabilizing proteins, for epithelial construction. Thus, as oviparity was replaced by viviparity, egg size was reduced, the germinal cytoplasm was retained, and yellow yolk was markedly reduced or lost in marsupials and eutherians. The white yolk was retained in monotremes and marsupials where blastocyst epithelial construction requires ECM support, and its appearance is heterochronously shifted to after compaction, when blastocyst formation and expansion occurs, in eutherian mammals.

Construction and characterization of phage display library: recognition of mouse serologically detected male (SDM) antigen.

Improvement of animal embryo sexing depends upon high-titer serologically detected male (SDM) antibody fragments. SDM sera collected from isogenic C57BL/7 female mice after inoculation with male spleen cells were characterized and used for construction of a recombinant Fab antibody library against SDM antigen, and used for analysis of the binding capacity and specificity to SDM antigen. The heavy-chain Fd and full-length light-chain kappa were amplified by RT-PCR from a mouse (#6) that'ed high-titer antiserum. The amplified product was inserted into the pComb3 vector followed by co-infections with the help phage VCSM 13 for construction of the phage library, which gave 1.5x10(7) colonies with the titer of 3.2x10(11) pfu/ml by a recombination rate of 80%. Sequence analysis of the PCR products of plasmid DNA of E5 clones showed that V(H) and V(kappa) had common characteristics shared by other known variable region of antibodies. The Fab antibody libraries against SDM antigen were enriched by three cycles of affinity enrichment with male spleen cells, and two cycles of non-specific absorption with female spleen cells. The ELISA results showed that 9 of 15 clones had binding capacity to the SDM antigen. This is the first report on a phage display library of SDM antigen. The mouse Fab antibody library could be used for identifying SDM antigen, and for the development of sex determination of early embryos in mammals.

[Cloning and analysis of phage Fab antibodies of mouse male specific antigen].

To clone mouse phage antibodies against H-Y antigen from a phage antibody library, three cycles of affinity enrichment of the mouse phage antibody library with male spleen cells and two cycles of nonspecific absorption with female spleen cells were performed. The presence of mouse Fab on the phage surface was determined by ELISA and sequence analysis. 9 of 15 strains can bind to male spleen cells with the specific activity. Recombination rate of the phage antibody library clones is 60%. Sequence analysis of the PCR products of plasmid DNA of E5 clones show VH and Vkappa had common characteristics shared by other known variable region of antibodies. The mouse phage Fab antibody could be used for identifying H-Y antigen, and for the development of sex determination of early embryos in mammals.

VAP1, with cystatin C motif, an oocyte protein encoded by a novel ovarian-specific gene during oogenesis in the common brushtail possum (Trichosurus vulpecula).

In the brushtail possum oocyte, vesicles accumulate in a polarized fashion at the vegetal pole and cytoplasm rich in mitochondria and containing the germinal vesicle comprise the animal pole. During cleavage to early blastocyst stages, animal pole cytoplasm locates to the cells of the embryonic hemisphere (pluriblast) and vegetal pole vesicular cytoplasm to cells of the abembryonic hemisphere (trophoblast). Previously identified 16 amino acid residues, associated with the vesicle-rich cytoplasm were used for molecular cloning and characterization of a vesicle associated protein, VAP1. The degenerate primer was used in a 3'RACE for vap1 gene cloning. The cDNA encoding VAP1 was 516 bp in length with no significant homologies and coded for 172 amino acid residues for the mature protein. The N-terminal domain of VAP1 showed a structural homology to the cysteine protease inhibitor, Cystatin. Gene expression studies during oogenesis revealed that vap1 had an ovary-specific, possibly oocyte-specific expression, which occurs during follicle formation and growth and in adult ovaries. Recombinant VAP1 fusion protein generated polyclonal antibodies in the mouse and in the brushtail possum.

Cloning and expression of a novel cDNA encoding shell coat protein, cp4, from the brushtail possum (Trichosurus vulpecula).

The marsupial conceptus is enclosed by several egg coats of evolutionary significance and unknown composition, of which the shell coat in mammals occurs only in marsupials and monotremes. Intact coats are vital to marsupial embryonic development. Towards a better understanding of the marsupial coat proteins, a cDNA sequence (cp4) encoding a shell coat protein was cloned from the brushtail possum. A cDNA library of a zygote stage uterus was screened using a deduced oligonucleotide sequence based on a partial amino acid sequence of the coat protein. This study has confirmed a single copy cp4 gene encoding a unique protein of 306 amino acids, although its N-terminus shares high sequence identity with the C-terminal half of the enzyme alpha-enolase. Using Northern blots, the expression of cp4 in adult tissues showed that cp4 transcript is restricted predominantly to the uterus with two stages of expression occurring in the gravid uterus at early cleavage and bilaminar stages, suggesting an important developmental role for CP4. Using RT-PCR, cp4-specific expression as represented by the 3'-end 400 bp was present in heart, liver, oviduct, and uterus. Uterine expression reflected the increase found with Northern blot except that expression was low at unilaminar and bilaminar stages.