Fam40b is required for lineage commitment of murine embryonic stem cells.

FAM40B (STRIP2) is a member of the striatin-interacting phosphatase and kinase (STRIPAK) complex that is involved in the regulation of various processes such as cell proliferation and differentiation. Its role for differentiation processes in embryonic stem cells (ESCs) is till now completely unknown. Short hairpin RNA (shRNA)-mediated silencing of Fam40b expression in ESCs and differentiating embryoid bodies (EBs) led to perturbed differentiation to embryonic germ layers and their derivatives including a complete abrogation of cardiomyogenesis. Pluripotency factors such as Nanog, Oct4 and Sox2 as well as epigenetic factors such as histone acetyltransferase type B (HAT1) and DNA (cytosine-5)-methyltransferase 3-ß (Dnmt3b) were highly upregulated in Fam40b knockdown EBs as compared with control and scrambled EBs. To examine the relevance of Fam40b for development in vivo, Fam40b was knocked down in developing zebrafish. Morpholino-mediated knockdown of Fam40b led to severe abnormalities of the cardiovascular system, including an impaired expression of ventricular myosin heavy chain (vmhc) and of cardiac myosin light chain 2 (cmlc2) in the heart. We identified the gene product of Fam40b in ESCs as a perinuclear and nucleolar protein with a molecular weight of 96 kDa. We conclude that the expression of Fam40b is essential for the lineage commitment of murine embryonic stem cells (mESCs) into differentiated somatic cells via mechanisms involving pluripotency and epigenetic networks.

Physical activity and alpha-lipoic acid modulate inflammatory response through changes in thiol redox status

Alpha-Lipoic acid (αLA), as an inductor of hydrogen peroxide (H2O2) and nitrogen oxide (NO) generation and modulator of thiol redox status, plays an important role in cell signalling pathways. The study was designed to observe the effect of AlphaLA on inflammatory response through changes in H2O2 and NO levels as well as thiol redox status. Sixteen physically active males were randomly assigned to one of two groups: placebo or αLA (1,200 mg d(-1) for 10 days prior to exercise). The exercise trial involved a 90-min run at 65% VO2max (0% gradient) followed by 15-min eccentric phase at 65% VO2max (-10% gradient). Blood samples were collected before the exercise trial and then again 20 min, 24, and 48 h after. AlphaLA significantly elevated H2O2 but reduced NO generation before or after exercise. Thiol redox status (GSHtotal-2GSSG/GSSG) increased by > 50% after αLA and exercise (ANOVA, P < 0.05) and correlated with changes in cytokines interleukin-6 (IL-6) (r = -0.478, P < 0.05) and IL-10 (r = -0.455, P < 0.05). This was caused by strong effect of αLA on GSSG concentration. αLA elevated IL-6 and IL-10 levels at 20 min after exercise and decreased in interleukin-1Beta and tumor necrosis factor Alpha before and after exercise. This enhanced the regeneration of injured muscles. Creatine kinase activity tended to lower values after αLA intake. The study suggests that the combination of intense exercise with α-lipoic acid intake might be useful to improve the skeletal muscle regeneration through changes in inflammatory response which are associated with H2O2 and NO generation as well as thiol redox status

Effect of shiitake (Lentinus edodes) extract on antioxidant and inflammatory response to prolonged eccentric exercise.

The shiitake (Lentinus edodes) extract is purported to have potent antioxidant, anti-inflammatory and regenerative properties due to presence of many bioactive compounds such as ergothioneine. This study was designed to assess the antioxidant and anti-inflammatory activity of shiitake extract in healthy men exposed to exercise-induced skeletal muscles damage. Subjects ingested shiitake mushroom extract (700 mg, two times per day) or placebo for 10 days prior to two separate exercise trials (crossover study). The exercise session involved 90 min run at 65% VO2max (0% gradient) and 15-min eccentric phase at 65% VO2max (-10% gradient). Subjects experienced creatine kinase (peak 461±206 IU/L) and leukocytes (peak 9.82 x 103/µL) elevations indicating muscle damage and inflammation. Exercise altered plasma IL-6 (peak 5.29±0.78 pg/mL), IL-10 (peak 24.75±6.22 pg/mL) and IL-1ß (peak 0.54±0.09 pg/mL) levels but did not affect tumour necrosis factor α (TNF-α) level relative to baseline. Shiitake extract did not demonstrate any effect on immune cells number and inflammatory mediators level, with the exception of IL-10. Thiol redox status (GSHtotal-2GSSG/GSSG) and niric oxide (NO) concentration increased after shiitake extract whereas H2O2 and 8-isoprostanes did not change. In conclusion, shiitake mushroom extract had no effect on markers of inflammation following prolonged eccentric exercise but demonstrated an antioxidant activity through the regulation of nitric oxide concentration and thiol redox status.

Physical activity and alpha-lipoic acid modulate inflammatory response through changes in thiol redox status.

α-Lipoic acid (αLA), as an inductor of hydrogen peroxide (H2O2) and nitrogen oxide (NO) generation and modulator of thiol redox status, plays an important role in cell signalling pathways. The study was designed to observe the effect of αLA on inflammatory response through changes in H2O2 and NO levels as well as thiol redox status. Sixteen physically active males were randomly assigned to one of two groups: placebo or αLA (1,200 mg d(-1) for 10 days prior to exercise). The exercise trial involved a 90-min run at 65% VO2max (0% gradient) followed by 15-min eccentric phase at 65% VO2max (-10% gradient). Blood samples were collected before the exercise trial and then again 20 min, 24, and 48 h after. αLA significantly elevated H2O2 but reduced NO generation before or after exercise. Thiol redox status (GSHtotal-2GSSG/GSSG) increased by >50% after αLA and exercise (ANOVA, P < 0.05) and correlated with changes in cytokines interleukin-6 (IL-6) (r = -0.478, P < 0.05) and IL-10 (r = -0.455, P < 0.05). This was caused by strong effect of αLA on GSSG concentration. αLA elevated IL-6 and IL-10 levels at 20 min after exercise and decreased in interleukin-1ß and tumor necrosis factor α before and after exercise. This enhanced the regeneration of injured muscles. Creatine kinase activity tended to lower values after αLA intake. The study suggests that the combination of intense exercise with α-lipoic acid intake might be useful to improve the skeletal muscle regeneration through changes in inflammatory response which are associated with H2O2 and NO generation as well as thiol redox status.

Cytokines and cellular inflammatory sequence in non-athletes after prolonged exercise.

AIM: The goal of the study was to clarify the sequence of cytokines and inflammatory cells in non-athletes performed an intense running exercise. METHODS: Sixteen young healthy men participated in the exercise trial that involved 90-min run at 65% VO2max. RESULTS: The plasma concentrations of anti-inflammatory cytokines IL-4, IL-6 and IL-10 increased immediately after exercise simultaneously with number of white blood cells. Between IL-6 and IL-10, and neutrophils the relationships were observed. The correlation value for IL-6 and neutrophils was 0.775 whereas for IL-10 and neutrophils was 0.506. The proinflammatory cytokines IL-1ß and TNFα were detected at 6 h after exercise and moderately correlated with monocytes count. The high level of proinflammatory cytokines, monocytes and creatine kinase (CK) remained until 48 h rest. The CK activity significantly correlated with IL-1ß (r=0.578) and TNFα (r=0.452), and also with monocytes count (r=0.439). CONCLUSION: The results have shown that: 1) exercise induces anti-inflammatory cytokines production first and then proinflammatory cytokines; and 2) prolonged proinflammatory response is closely related with muscle damage present.

Nuclear transport receptor karyopherin-α2 promotes malignant breast cancer phenotypes in vitro.

Tumorigenesis and tumor progression are associated with dysfunction of the nuclear transport machinery at the level of import and export receptors (karyopherins). Recent studies have shown that the nuclear import factor karyopherin-α2 (KPNA2) is a novel prognostic marker for poor prognosis in human breast cancer. Based on the well-defined hallmarks of cancer progression, we performed a detailed in vitro characterization of the phenotypic effects caused by KPNA2 overexpression and KPNA2 silencing in benign and malignant human breast cells. KPNA2 overexpression clearly increased proliferation of MCF7 tumor cells and further led to a reduction of cell-matrix adhesion in benign MCF10A cells, whereas cell migration was significantly increased (P<0.0001) in both tumor models. Remarkably, these individual effects of KPNA2 overexpression on proliferation, cell-matrix adhesion and migration resulted in an increased colony spreading of benign MCF10A breast cells and malignant MCF7 tumor cells (P<0.001), which is a hallmark of cancer progression. Conversely, RNA interference-mediated KPNA2 silencing caused a complete inhibition of MCF7 tumor cell proliferation and migration (P<0.0001). In addition, in these experiments apoptosis was increased (P<0.05) and formation of tumor cell colonies was reduced (P<0.01). Thus, KPNA2 overexpression provoked increased aggressiveness of malignant MCF7 breast tumor cells and induced a shift in benign MCF10A breast cells toward a malignant breast cancer phenotype. In conclusion, we demonstrate for the first time in experimental tumor models that forced KPNA2 expression drives malignant features relevant for breast cancer progression, while its silencing is required for the remission of those progressive phenotypes. This study gives clear evidence that KPNA2 acts as a novel oncogenic factor in human breast cancer, in vitro.

Changes of muscle-derived cytokines in relation to thiol redox status and reactive oxygen and nitrogen species.

The aim of this study was to compare the levels of the plasma muscle-derived cytokines (myokines) and reactive oxygen and nitrogen species (RONS) after muscle damage triggered by different exercises, and to demonstrate the relationships between RONS, thiol redox status and myokines. Sixteen young men participated in a 90-min run at 65% VO2max (Ex.1) or 90-min run at 65% VO2max finished with a 15-min eccentric phase (Ex.2, downhill running). Plasma samples were collected before and at 20 min, 24 h and 48 h after exercise. The exercise trials significantly elevated the concentrations of plasma hydrogen peroxide (H2O2) and 8-isoprostane at 20 min rest. Myokines IL-6 and IL-10 increased at 20 min rest while IL-1ß and TNFα increased at 24 h rest following both running. Ex.2 caused a significant increase in nitric oxide (NO), IL-6, IL-10 and oxidized glutathione (GSSG) levels. Thiol redox status (GSH(total)-2GSSG/GSSG) decreased by about 30% after Ex.2 as compared to Ex.1. H2O2) and NO directly correlated with IL-6, IL-10, IL-1ß, TNFα and glutathione. These results show that eccentric work is an important factor that enhances the production of RONS and muscle-derived cytokines, and that there is a possible participation of thiol redox status in the release of myokines to blood.

Homologues of c-hairy1 (her9) and lunatic fringe in zebrafish are expressed in the developing central nervous system, but not in the presomitic mesoderm.

A number of genes that are involved in somitogenesis in vertebrates are cyclically expressed in the presomitic mesoderm. These include homologues of the Drosophila genes fringe and hairy. We have analysed here two genes that belong to these classes in the zebrafish, namely the apparent orthologues of lunatic fringe (l-fng) and of c-hairy1 (called her9). However, unlike the respective mouse and chicken genes, they are not expressed cyclically in the presomitic mesoderm. Instead, both genes are mainly expressed in the central nervous system. her9 is predominantly expressed in the fore- and midbrain, and transiently in the hindbrain. Thus, the previously identified and only very distantly related her1 gene of zebrafish has more similarities to the expression of the c-hairy1 gene than its apparent orthologue her9, indicating that sequence similarity and similarity of function are not necessarily linked in this case. l-fng expression is found in alternating pre-rhombomeres, comparable to the equivalent mouse gene expression and in the anterior compartments of the mature somites, which was also shown for the chicken l-fng gene. The latter expression indicates that it might be involved in boundary definition and cell fate decision processes, rather than in pre-patterning of the somites. Interestingly, a similar role has previously been inferred for the grasshopper homologue of l-fng. This suggests that the function of l-fng in boundary definition of the somites might be ancestral, while its recruitment to the pre-patterning process of the somites might be a derived feature in higher vertebrates.

Electrospray tandem mass spectrometry of lexitropsins.

Several compounds, representative of the class of lexitropsins, were analyzed by electrospray tandem mass spectrometry. The study of the fragmentations of the protonated molecular species ([M + H](+)) and of selected fragment ions allowed proposals for the main fragmentation pathways of compounds of this type. The interpretation of the fragmentation pathways of these compounds was complicated because of intramolecular hydrogen migration. In order to better understand the fragmentation pathways, the MS/MS/MS spectra of several compounds, and the MS/MS and MS/MS/MS spectra of the deuterated compounds, were obtained. Accurate mass measurements helped elucidate the structures of smaller fragment ions. Low-energy collision-induced decomposition (CID) tandem mass spectrometry of lexitropsins with electrospray ionization has proven to be a good method for the structural characterization and identification of this class of compounds. Main fragmentation pathways occur by cleavage of the peptide bond followed by the elimination of the substituted pyrrole ring, and their elucidation will facilitate structural characterization of new lexitropsins.

Generating autogenous cartilage on the basis of subperichondrial grafts of demineralized bone matrix with local stimulation by growth factors.

Cartilage tissue acquired de novo is a very attractive material for the surgical reconstruction of joint surfaces, trachea, and maxillofacial elements. One of the primary challenges for tissue engineering is to determine the procedures that lead to the creation of material meeting the established qualitative and quantitative criteria. The goal of this work was to determine whether and how growth factors (IGF-1 and FGF) and ethyl alcohol administered locally affect the course and final outcome of the chondrogenetic process under in vitro conditions in New Zealand rabbits. In order to generate cartilage, a collagen scaffold (demineralized ZMK bone matrix) was used, wrapped in a pedunculated flap of perichondrium (from the rabbit's ear), which, beginning on the 3rd day after the operation, was injected with growth factors every 3 days. Grafts were collected in the 3rd and 6th week after the ZMK implantation, and the silvers made from them were stained for the presence of collagen II, collagen I, and macrophages, and analyzed morphometrically. It was found that the application of growth factors only slighty, intensified the synthesis of collagen II, and had no effect on the degree of macrophage infiltration or collagen I contents, while the numerous injections exerted a negative impact on the architecture of the newly-formed tissue and contributed to an increased number of complications (hematomas, infections).

Tissue engineering in reconstructive surgery of bone and cartilage.

Molecular research has Bern the subject of considerable interest in recent years. The same can also be said for tissue engineering, which has ushered us into a world previously accessible only in science fiction. The possibility of creating human tissue opens the road for reconstructive surgery using biologically matched grafts. Some of the tissue engineering methods that pertain to orthopedics have already found application in the clinic. Among these are operations to reconstruct defects of joint cartilage, based on the in vitro multiplication of chondrocytes isolated from cartilage fragments previously collected arthroscopically. By applying tissue engineering technology we will soon be able to culture the patient's own bone tissue needed to fill defects in the bone bed. At some point in the not too distant future we will be able to graft entire joint ends, instead of the joint endoprosthesoplastic procedures currently in use. Tissue engineering, like every new field of science, prompts emotional reactions not only in medical circles, but also in many social groups. Let us hoper for fulfillment of the anticipation that a way will be found to overcome disabilities involving the locomotor apparatus.
The authors, who come from varying theoretical and clinical settings, present a short history of tissue engineering, based on their own experience and the available literature, the current possibilities to use tissue engineering in the reconstruction of bone and cartilage, and the prospects for development in the field in the near future.