Cellular reprogramming of somatic cells

The process of 'cell reprogramming' can be achieved by somatic cell nuclear transfer, cell fusion with embryonic stem cells, exposure to stem cell extracts, or by inducing pluripotentcy mediated by defined factors giving rise to what are termed induced pluripotent stem cells. More recently, the fate of a somatic cell can be directly induced to uptake other cell fates, termed lineage-specific reprogramming, without the need to de-differentiate the cells to a pluripotent state. In this review we will describe the different methods of reprogramming somatic cells.     

Norrin immunolocalization and its possible functions in rat endometrium during the estrus cycle and early pregnancy

Mutations in Norrie Disease Pseudoglioma (NDP) gene cause serious sight loss, deafness and mental retardation in Norrie disease patients via the impairment of angiogenesis. Since norrin is a Wnt pathway ligand, it could function in several tissues other than eye and nervous systems. Therefore, the aim of the present study was to determine the possible function of norrin in angiogenesis, cellular differentiation in stroma and in decidua and the survival of those cells using immunofluorescent labeling. While norrin had a uniform distribution in stroma and in blood vessels, it had a strong expression in luminal and glandular epithelia during the estrus cycle. Norrin had strong immunolocalization in the antimesometrial decidual reaction zone on day 7 of gestation, whereas it had a decreased expression in the mesometrial uterine luminal epithelium along with an increased localization in blood vessels and decidual cells of the same region on day 8 of gestation. As from day 9 of gestation, norrin demonstrated rather strong expression in the decidual cells and blood vessels of the mesometrial region in which the chorioallantoic placenta was going to develop. In all periods studied, norrin had rather weak expression in the primary decidual zone surrounding the embryo. Findings of the present study suggested that norrin might regulate the decidual reaction and the placental angiogenesis along with the survival and the differentiation of luminal and glandular epithelial and decidual cells in rats. In addition, it could play indirect important roles in the control of trophoblastic invasion and the programmed cell death.     

A two-stage pretreatment of seedlings improves adventitious shoot regeneration in sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.)

The effects of a two-stage pretreatment of seedlings on the subsequent shoot regeneration capacity were investigated. Pretreated seedlings were obtained by germinating seeds on three different germination media and then further culturing on six different growth media. Lamina and petiole explants of two sugar beet (Beta vulgaris L.) breeding lines were then excised from the pretreated seedlings and cultured on five different shoot regeneration media. In both breeding lines, petiole explants produced significantly more shoots than lamina explants with higher frequencies of organogenic capacities; petiole explants of the lines M1195 and ELK345 produced a mean of 2.1 and 2.7 shoots per explant while their lamina explants produced 1.5 and 2.2 shoots per explant, respectively. A genotypic variation was evident as the line ELK345 was more productive for shoot development from both types of explants. In overall comparisons of different germination, growth and regeneration media, germination medium was most effective when supplemented with 0.5 mg/l 6-benzyladenine (BA) while both growth and regeneration media were most productive when contained a combination of 0.25 mg/l BA and 0.10 mg/l indole-3-butyric acid (IBA). Of all the treatments tested, the highest mean number of shoots per explant (8.3 shoots) and frequency of organogenic explants (75.6%) were obtained on regeneration medium supplemented with 0.25 mg/l BA and 0.10 mg/l IBA when petiole explants of the line ELK345 were excised from the seedlings that had been germinated on medium containing 0.5 mg/l BA followed by further growth on medium containing 0.25 mg/l BA and 0.10 mg/l IBA.     

The effect of extracellular matrix on the differentiation of mouse embryonic stem cells

Embryonic stem cells (ESCs) are promising research materials to investigate cell fate determination since they have the capability to differentiate. Stem cell differentiation has been extensively studied with various microenvironment mimicking structures to modify cellular dynamics associated with the cell-extracellular matrix (ECM) interactions and cell-cell communications. In the current study, our aim was to determine the effect of microenvironmental proteins with different concentrations on the capacity and differentiation capability of mouse ESCs (mESCs), combining the biochemical assays, imaging techniques, Fourier transform infrared (FTIR) spectroscopy, and unsupervised multivariate analysis. Based on our data, coating the surface of mESCs with Matrigel, used as an acellular matrix substrate, resulted in morphological and biochemical changes. mESCs exhibited alterations in their phenotype after growing on the Matrigel-coated surfaces, including their differentiation capacity, cell cycle phase pattern, membrane fluidity, and metabolic activities. In conclusion, mESCs can be stimulated physiologically, chemically, or mechanically to convert them a new phenotype. Thus, identification of ESCs’ behavior in the acellular microenvironment could be vital to elucidate the mechanism of diseases. It might also be promising to control the cell fate in the field of tissue engineering.     

Pancreatic Endocrine and Exocrine Function in Children following Near-Total Pancreatectomy for Diffuse Congenital Hyperinsulinism

Context

Congenital hyperinsulinism (CHI), the commonest cause of persistent hypoglycaemia, has two main histological subtypes: diffuse and focal. Diffuse CHI, if medically unresponsive, is managed with near-total pancreatectomy. Post-pancreatectomy, in addition to persistent hypoglycaemia, there is a very high risk of diabetes mellitus and pancreatic exocrine insufficiency.

Setting

International referral centre for the management of CHI.

Patients

Medically unresponsive diffuse CHI patients managed with near-total pancreatectomy between 1994 and 2012.

Intervention

Near-total pancreatectomy.

Main Outcome Measures

Persistent hypoglycaemia post near-total pancreatectomy, insulin-dependent diabetes mellitus, clinical and biochemical (faecal elastase 1) pancreatic exocrine insufficiency.

Results

Of more than 300 patients with CHI managed during this time period, 45 children had medically unresponsive diffuse disease and were managed with near-total pancreatectomy. After near-total pancreatectomy, 60% of children had persistent hypoglycaemia requiring medical interventions. The incidence of insulin dependent diabetes mellitus was 96% at 11 years after surgery. Thirty-two patients (72%) had biochemical evidence of severe pancreatic exocrine insufficiency (Faecal elastase 1<100 µg/g). Clinical exocrine insufficiency was observed in 22 (49%) patients. No statistically significant difference in weight and height standard deviation score (SDS) was found between untreated subclinical pancreatic exocrine insufficiency patients and treated clinical pancreatic exocrine insufficiency patients.

Conclusions

The outcome of diffuse CHI patients after near-total pancreatectomy is very unsatisfactory. The incidence of persistent hypoglycaemia and insulin-dependent diabetes mellitus is very high. The presence of clinical rather than biochemical pancreatic exocrine insufficiency should inform decisions about pancreatic enzyme supplementation.     

Characterization and inhibition studies of an α-carbonic anhydrase from the endangered sturgeon species Acipenser gueldenstaedti

An α-carbonic anhydrase (CA, EC 4.2.1.1) was purified and characterized kinetically from erythrocytes of the sturgeon Acipenser gueldenstaedti, an endangered species. The sturgeon enzyme (AgCA) showed kinetic parameters for the CO(2) hydration reaction comparable with those of the human erythrocytes enzyme hCA II, being a highly active enzyme, whereas its esterase activity with 4-nitrophenyl acetate as substrate was lower. Sulphonamide inhibitors (acetazolamide, sulphanilamide) strongly inhibited AgCA, whereas metal ions (Ag(+), Zn(2+), Cu(2+) and Co(2+)) were weak, millimolar inhibitors. Several widely used pesticides (2,4-dichlorophenol, dithiocarbamates, parathion and carbaryl) were also assayed as inhibitors of this enzyme. The dithiocarbamates were low micromolar AgCA inhibitors (IC(50) of 16-18 μM), whereas the other pesticides inhibited the enzyme with IC(50)s in the range of 102-398 μM. The wide use of dithiocarbamate pesticides may be one of the factors enhancing the vulnerability of this sturgeon species to pollutants.     

Production of methionine γ- lyase in recombinant Citrobacter freundii bearing the hemoglobin gene

The production of antileukemic enzyme methionine γ-lyase (MGL) in distinctly related bacteria, Citrobacter freundii and in their recombinants expressing the Vitresocilla hemoglobin (VHb) has been studied. This study concerns the potential of Citrobacter freundii expressing the Vitreoscilla hemoglobin gene (vgb) for the methionine γ- liyase production. Methionine γ- liyase production by Citrobacter freundii and its vgb(-) and vgb(+) bearing recombinant strain was studied in shake-flasks under 200 rpm agitation, culture medium and 30 °C in a time-course manner. The vgb(+) and especially the carbon type had a dramatic effect on methionine γ- liyase production. The vgb(+) strain of C. freundii had about 2-fold and 3.1-fold higher levels of MGL than the host and vgb(-) strain, respectively.