A Novel Source of Cultured Podocytes

Amniotic fluid is in continuity with multiple developing organ systems, including the kidney. Committed, but still stem-like cells from these organs may thus appear in amniotic fluid. We report having established for the first time a stem-like cell population derived from human amniotic fluid and possessing characteristics of podocyte precursors. Using a method of triple positive selection we obtained a population of cells (hAKPC-P) that can be propagated in vitro for many passages without immortalization or genetic manipulation. Under specific culture conditions, these cells can be differentiated to mature podocytes. In this work we compared these cells with conditionally immortalized podocytes, the current gold standard for in vitro studies. After in vitro differentiation, both cell lines have similar expression of the major podocyte proteins, such as nephrin and type IV collagen, that are characteristic of mature functional podocytes. In addition, differentiated hAKPC-P respond to angiotensin II and the podocyte toxin, puromycin aminonucleoside, in a way typical of podocytes. In contrast to immortalized cells, hAKPC-P have a more nearly normal cell cycle regulation and a pronounced developmental pattern of specific protein expression, suggesting their suitability for studies of podocyte development for the first time in vitro. These novel progenitor cells appear to have several distinct advantages for studies of podocyte cell biology and potentially for translational therapies.     

Priming xylem for stress recovery depends on coordinated activity of sugar metabolic pathways and changes in xylem sap pH

Some plant species are capable of significant reduction of xylem embolism during recovery from drought despite stem water potential remains negative. However, the functional biology underlying this process is elusive. We subjected poplar trees to drought stress followed by a period of recovery. Water potential, hydraulic conductivity, gas exchange, xylem sap pH, and carbohydrate content in sap and woody stems were monitored in combination with an analysis of carbohydrate metabolism, enzyme activity, and expression of genes involved in sugar metabolic and transport pathways. Drought resulted in an alteration of differential partitioning between starch and soluble sugars. Upon stress, an increase in the starch degradation rate and the overexpression of sugar symporter genes promoted the efflux of disaccharides (mostly maltose and sucrose) to the apoplast. In turn, the efflux activity of the sugar‐proton cotransporters caused a drop in xylem pH. The newly acidic environment induced the activity of apoplastic invertases leading to the accumulation of monosaccharides in the apoplast, thus providing the main osmoticum necessary for recovery. During drought and recovery, a complex network of coordinated molecular and biochemical signals was activated at the interface between xylem and parenchyma cells that appeared to prime the xylem for hydraulic recovery.     

Effect of extracellular matrix proteins on in vitro testosterone production by rat Leydig cells

The aim of this study was to detect the effect of extracellular matrix (ECM) proteins on rat Leydig cell shape, adhesion, expression of integrin subunits and testosterone production, in vitro. Leydig cells isolated from adult rats were cultured on plates uncoated or coated with different concentrations of laminin-1, fibronectin, or type IV collagen in the presence or absence of hCG for 3 or 24 hr. A significant increase of cell adhesion and of alpha3, alpha5, and beta1 integrin subunit expression was observed when cells were cultured on ECM proteins, compared to those grown on uncoated plates. Leydig cells cultured on glass coverslips coated with ECM proteins for 24 hr exhibited elongated shapes with long cell processes (spreading), while cells cultured on uncoated plates showed few cell processes. A significant decrease in testosterone production was observed when basal and hCG-stimulated Leydig cells were cultured for 3 or 24 hr on plates coated with type IV collagen (12 and 24 microg/cm(2)) compared to uncoated plates. A significant though a slighter decrease in testosterone production was also observed in cells cultured on plates coated with fibronectin (12 and 24 microg/cm(2)), compared to uncoated plates. Laminin-1 did not modify testosterone production under basal or hCG stimulated conditions. These results suggest that ECM proteins are able to modulate Leydig cell steroidogenesis, in vitro.     

Embryo development in vitro of cat oocytes cryopreserved at different maturation stages

The purpose of this study was to evaluate the ability of cat oocytes, at different stages of maturation, to survive after cryopreservation and to assess their subsequent development following IVM and IVF. In the initial toxicity trial, immature oocytes were exposed to different concentrations of DMSO and ethylene glycol (EG). Resumption of meiosis and metaphase II were evaluated after removal of the cryoprotectant and IVM. The highest rates of resumption of meiosis (51.4%) were achieved after exposure to 1.5 mol l(-1) of cryoprotectants, and no difference was observed with control oocytes. Metaphase II was obtained in 25.7% (P<0.01) and 22.9% (P<0.005) of oocytes exposed to 1.5 mol l(-1) of DMSO and ethylene glycol, although at lower rates than in control oocytes (54.4%). On the basis of this finding, 1.5 mol l(-1) of cryoprotectant was chosen for freezing cat oocytes at the germinal vesicle stage (immature) or at metaphase II stage (mature). Post-thaw viability was assessed by the evaluation of the embryo development in vitro. After fertilization, mature oocytes frozen in ethylene glycol cleaved in better proportions (38.7%) than immature oocytes (6.8%, P<0.001), and no differences were observed in the cleavage rate of oocytes frozen at different maturation stages with DMSO (immature 12.8%; mature 14.1%). Embryonic development beyond the 8-cell stage was obtained only when mature oocytes were frozen with ethylene glycol (11.3%). This study suggests that cryopreserved cat oocytes can be fertilized successfully and that their development in vitro is enhanced when mature oocytes are frozen with ethylene glycol. The stage of maturation may be a key element in improving cat oocyte cryopreservation.     

Expression and function of the homeodomain-containing protein Hex in thyroid cells

The homeodomain-containing protein Hex (also named Prh) is expressed in primitive endoderm (during the early phases of development), in some endoderm-derived tissues and in endothelial and hematopoietic precursors. Hex expression is extinguished during terminal differentiation of endothelial and hematopoietic cells as well as in adult lung. Previous investigations have demonstrated that Hex is expressed during early thyroid gland development. No information has been reported on Hex expression in adult thyroid gland or on the function of this protein in follicular thyroid cells. These issues represent the focus of the present study. We demonstrate that Hex mRNA is present in rat and human adult thyroid gland as well as in differentiated follicular thyroid cell lines. In FRTL-5 cells TSH reduces Hex expression. In thyroid cell lines transformed by several oncogenes Hex expression is completely abolished. By using co-transfection assays we demonstrate that Hex is a repressor of the thyroglobulin promoter and that it is able to abolish the activating effects of both TTF-1 and Pax8. These data would suggest that Hex may play an important role in thyroid cell differentiation. Protein–DNA interaction experiments indicate that Hex is able to bind sites of the thyroglobulin promoter containing either the core sequence 5′-TAAT-3′ or 5′-CAAG-3′. The DNA binding specificity of the Hex homeodomain, therefore, is more ‘relaxed’ than that observed in the majority of other homeodomains.     

H-Ferritin-Regulated MicroRNAs Modulate Gene Expression in K562 Cells

In a previous study, we showed that the silencing of the heavy subunit (FHC) offerritin, the central iron storage molecule in the cell, is accompanied by a modification in global gene expression. In this work, we explored whether different FHC amounts might modulate miRNA expression levels in K562 cells and studied the impact of miRNAs in gene expression profile modifications. To this aim, we performed a miRNA-mRNA integrative analysis in K562 silenced for FHC (K562^shFHC) comparing it with K562 transduced with scrambled RNA (K562^shRNA). Four miRNAs, namely hsa-let-7g, hsa-let-7f, hsa-let-7i and hsa-miR-125b, were significantly up-regulated in silenced cells. The remarkable down-regulation of these miRNAs, following FHC expression rescue, supports a specific relation between FHC silencing and miRNA-modulation. The integration of target predictions with miRNA and gene expression profiles led to the identification of a regulatory network which includes the miRNAs up-regulated by FHC silencing, as well as91 down-regulated putative target genes. These genes were further classified in 9 networks; the highest scoring network, “Cell Death and Survival, Hematological System Development and Function, Hematopoiesis”, is composed by 18 focus molecules including RAF1 and ERK1/2. We confirmed that, following FHC silencing, ERK1/2 phosphorylation is severely impaired and that RAF1 mRNA is significantly down-regulated. Taken all together, our data indicate that, in our experimental model, FHC silencing may affect RAF1/pERK1/2 levels through the modulation of a specific set of miRNAs and add new insights in to the relationship among iron homeostasis and miRNAs.     

TLR9-independent activation of B lymphocytes by bacterial DNA

The intracellular Toll-like receptor 9 (TLR9) is unique in its ability to recognize single-stranded DNA unmethylated at CpG motifs. Work from this laboratory showed that plasmid DNA is spontaneously internalized in B lymphocytes. This event is followed by the upregulation of costimulatory molecules and the acquisition of antigen presenting function by these cells. However, it is not known whether this phenomenon depends on TLR9. Because of the relevant role played by DNA-based drugs in immunotherapy and vaccination, and the central role of TLR9 signaling by CpG motifs, we decided to investigate whether signaling through TLR9 is a prerequisite for spontaneous transgenesis of lymphocytes. Here we found that transgene expression and upregulation of CD40 and CD86 costimulatory molecules was not inhibited by chloroquine treatment. Spontaneous transgenesis also occurred in B lymphocytes from TLR9-/- mice, and the injection of TLR9-/- transgenic B lymphocytes in C57Bl/6 mice induced both CD4 and CD8 T cell responses comparable to those induced by wild-type B lymphocytes. Collectively, these results suggest that plasmid DNA activates mammalian B lymphocytes through a TLR9 independent pathway.