A Pan-ALDH1A Inhibitor Induces Necroptosis in Ovarian Cancer Stem-like Cells

1. Download high-res image (179KB)
2. Download full-size image

     

PyMINEr Finds Gene and Autocrine-Paracrine Networks from Human Islet scRNA-Seq

1. Download high-res image (260KB)
2. Download full-size image

     

Comparison of insect kinin analogs with cis-peptide bond motif 4-aminopyroglutamate identifies optimal stereochemistry for diuretic activity

The insect kinins are present in a wide variety of insects and function as potent diuretic peptides, though they are subject to rapid degradation by internal peptidases. Insect kinin analogs incorporating stereochemical variants of (2S,4S)-4-aminopyroglutamate (APy), a cis-peptide bond motif, demonstrate significant activity in a cricket diuretic assay. Insect kinin analogs containing (2R,4R)-APy, (2S,4R)-APy and (2S,4S)-APy are essentially equipotent on an insect diuretic assay, with EC(50) values of about 10(-7)M, whereas the (2R,4S)-APy analog is at least 10-fold more potent (EC(50) = 7 x 10(-9)M). Conformational studies in aqueous solution indicate that the (2R,4S)-APy analog is considerably more flexible than the other three variants, which may explain its greater potency. The work identifies the optimal stereochemistry for the APy scaffold with which to design biostable, peptidomimetic analogs with the potential to disrupt critical insect kinin-regulated processes in insects.     

Dynamic analysis of GS-NS0 cells producing a recombinant monoclonal antibody during fed-batch culture

In this study we have analyzed the dynamic covariation of the mammalian cell proteome with respect to functional phenotype during fed-batch culture of NS0 murine myeloma cells producing a recombinant IgG(4) monoclonal antibody. GS-NS0 cells were cultured in duplicate 10 L bioreactors (36.5 degrees C, 15% DOT, pH 7.0) for 335 h and supplemented with a continuous feed stream after 120 h. Cell-specific growth rate declined continuously after 72 h of culture. Cell-specific recombinant monoclonal antibody production rate (qP) varied sixfold through culture. Whilst qP correlated with relative recombinant heavy chain mRNA abundance up to 216 h, qP subsequently declined, independent of recombinant heavy chain or light chain mRNA abundance. GS-NS0 cultures were sampled at 48 h intervals between 24 and 264 h of culture for proteomic analyses. Total protein abundance and nascent polypeptide synthesis was determined by 2D PAGE of unlabeled proteins visualized by SYPRO Ruby and autoradiography of (35)S-labeled polypeptides, respectively. Covariation of nascent polypeptide synthesis and abundance with biomass-specific cell growth, glucose and glutamate consumption, lactate and Mab production rates were then examined using two partial least squares regression models. Most changes in polypeptide synthesis or abundance for proteins previously identified by mass spectrometry were positively correlated with biomass-specific growth rate. We conclude that the substantial transitions in cell physiology and qP that occur during culture utilize a relatively constant complement of the most abundant host cell machines that vary primarily with respect to induced changes in cell growth rate.     

The inhibition of T-cells proliferation by mouse mesenchymal stem cells through the induction of p16INK4A-cyclin D1/cdk4 and p21waf1, p27kip1-cyclin E/cdk2 pathways

Mesenchymal stem cells (MSCs) have been shown to down-regulate T-cell responses. However, the mechanisms underlying remain unknown. In this study, we report that BALB/c bone marrow-derived MSCs inhibit the proliferation of allogeneic T-cells in mixed lymphocyte reactions (MLR), This inhibition is dependent on cell-cell contact, and do not induce apoptosis. Furthermore, cell-cycle analyses reveal that T-cells, in the presence of MSCs, are arrested in the G0/G1 phase through. The blockage of phosphorylation of retinoblastoma protein (Rb), mediated by the p16(INK4A)-cyclin D1/cdk4 complex and p21(waf1), p27(kip1)-cyclin E/cdk2 complex pathway. Our results suggest that MSCs may perform a crucial function in the maintenance of immune homeostasis, via direct regulation of the clonal expansion of activated T-cells. The novel T-cell regulatory mechanism exhibited by MSCs may prove useful in a variety of therapeutic applications.     

Age-, gender-, and species-dependent mutagenicity in T cells of mice and rats exposed by inhalation to 1,3-butadiene

Experiments were performed: (i) to investigate potential age- and gender-dependent differences in mutagenic responses in T cells following exposures of B6C3F1 mice and F344 rats by inhalation for 2 weeks to 0 or 1250 ppm butadiene (BD), and (ii) to determine if exposures for 2 weeks to 62.5 ppm BD produce a mutagenic effect in female rats. To evaluate the effect of age on mutagenic response, mutant manifestation curves for splenic T cells of female mice exposed at 8-9 weeks of age were defined by measuring Hprt mutant frequencies (MFs) at multiple time points after BD exposure using a T cell cloning assay and comparing the resulting mutagenic potency estimate (calculated as the difference of areas under the mutant manifestation curves of treated versus control animals) to that reported for female mice exposed to BD in the same fashion beginning at 4-5 weeks of age. The shapes of the mutant T cell manifestation curves for spleens were different [e.g., the maximum BD-induced MFs in older mice (8.0+/-1.0 [S.D.]x10(-6)) and younger mice (17.8+/-6.1 x 10(-6)) were observed at 8 and 5 weeks post-exposure, respectively], but the mutagenic burden was the same for both age groups. To assess the effect of gender on mutagenic response, female and male rodents were exposed to BD at 4-5 weeks of age and Hprt MFs were measured when maximum MFs are expected to occur post-exposure. The resulting data demonstrated that the pattern for mutagenic susceptibility from high-level BD exposure is female mice>male mice>female rats>male rats. Exposures of female rats to 62.5 ppm BD caused a minor but significant mutagenic response compared with controls (n=16/group; P=0.03). These results help explain part of the differing outcomes/interpretations of data in earlier Hprt mutation studies in BD-exposed rodents.     

Prostaglandin E2 regulates melanocyte dendrite formation through activation of PKCzeta

Prostaglandins are lipid signaling intermediates released by keratinocytes in response to ultraviolet irradiation (UVR) in the skin. The main prostaglandin released following UVR is PGE(2), a ligand for 4 related G-protein-coupled receptors (EP(1), EP(2), EP(3) and EP(4)). Our previous work established that PGE(2) stimulates melanocyte dendrite formation through activation of the EP(1) and EP(3) receptors. The purpose of the present report is to define the signaling intermediates involved in EP(1)- and EP(3)-dependent dendrite formation in human melanocytes. We recently showed that activation of the atypical PKCzeta isoform stimulates melanocyte dendricity in response to treatment with lysophosphatidylcholine. We therefore examined the potential contribution of PKCzeta activation on EP(1)- and EP(3)-dependent dendrite formation in melanocytes. Stimulation of the EP(1) and EP(3) receptors by selective agonists activated PKCzeta, and inhibition of PKCzeta activation abrogated EP(1)- and EP(3)-receptor-mediated melanocyte dendricity. Because of the importance of Rho-GTP binding proteins in the regulation of melanocyte dendricity, we also examined the effect of EP(1) and EP(3) receptor activation on Rac and Rho activity. Neither Rac nor Rho was activated upon treatment with EP(1,3)-receptor agonists. We show that melanocytes express only the EP(3A1) isoform, but not the EP(3B) receptor isoform, previously associated with Rho activation, consistent with a lack of Rho stimulation by EP(3) agonists. Our data suggest that PKCzeta activation plays a predominant role in regulation of PGE(2)-dependent melanocyte dendricity.     

A highly conserved Wnt-dependent TCF4 binding site within the proximal enhancer of the anti-myogenic Msx1 gene supports expression within Pax3-expressing limb bud muscle precursor cells

The product of the Msx1 gene is a potent inhibitor of muscle differentiation. Msx1 is expressed in muscle precursor cells of the limb bud that also express Pax3. It is thought that Msx1 may facilitate distal migration by delaying myogenesis in these cells. Despite the role played by Msx1 in inhibiting muscle differentiation, nothing is known of the mechanisms that support the expression of the Msx1 gene within limb bud muscle precursor cells. In the present study we have used a combination of comparative genomics, mouse transgenic analysis, in situ hybridisation and immunohistochemistry to identify a highly conserved and tissue-specific regulatory sub-domain within the previously characterised Msx1 gene proximal enhancer element that supports the expression of the Msx1 gene in Pax3-expressing mouse limb pre-muscle masses. Furthermore, using a combination of in situ hybridisation, in vivo ChIP assay and transgenic explant culture analysis we provide evidence that Msx1 expression in limb bud muscle precursor cells is dependent on the canonical Wnt/TCF signalling pathway that is important in muscle shape formation. The results of these studies provide evidence of a mechanistic link between the Wnt/TCF and the Msx1/Pax3/MyoD pathways within limb bud muscle precursor cells.     

Essential functions of Alk3 during AV cushion morphogenesis in mouse embryonic hearts

Accumulated evidence has suggested that BMP pathways play critical roles during mammalian cardiogenesis and impairment of BMP signaling may contribute to human congenital heart diseases (CHDs), which are the leading cause of infant morbidity and mortality. Alk3 encodes a BMP specific type I receptor expressed in mouse embryonic hearts. To reveal functions of Alk3 during atrioventricular (AV) cushion morphogenesis and to overcome the early lethality of Alk3(-/-) embryos, we applied a Cre/loxp approach to specifically inactivate Alk3 in the endothelium/endocardium. Our studies showed that endocardial depletion of Alk3 severely impairs epithelium-mesenchymal-transformation (EMT) in the atrioventricular canal (AVC) region; the number of mesenchymal cells formed in Tie1-Cre;Alk3(loxp/loxp) embryos was reduced to only approximately 20% of the normal level from both in vivo section studies and in vitro explant assays. We showed, for the first time, that in addition to its functions on mesenchyme formation, Alk3 is also required for the normal growth/survival of AV cushion mesenchymal cells. Functions of Alk3 are accomplished through regulating expression/activation/subcellular localization of multiple downstream genes including Smads and cell-cycle regulators. Taken together, our study supports the notion that Alk3-mediated BMP signaling in AV endocardial/mesenchymal cells plays a central role during cushion morphogenesis.     

Single-gene detection and karyotyping using small-target fluorescence in situ hybridization on maize somatic chromosomes

Combined with a system for identifying each of the chromosomes in a genome, visualizing the location of individual genetic loci by fluorescence in situ hybridization (FISH) would aid in assembling physical and genetic maps. Previously, large genomic clones have been successfully used as FISH probes onto somatic chromosomes but this approach is complicated in species with abundant repetitive elements. In this study, repeat-free portions of sequences that were anchored to particular chromosomes including genes, gene clusters, large cDNAs, and portions of BACs obtained from public databases were used to label the corresponding physical location using FISH. A collection of probes that includes at least one marker on each chromosome in the maize complement was assembled, allowing a small-target karyotyping system to be developed. This set provides the foundation onto which additional loci could be added to strengthen further the ability to perform chromosomal identification in maize and its relatives. The probes were demonstrated to produce signals in several wild relatives of maize, including Zea luxurians, Z. diploperennis, and Tripsacum dactyloides.