Neurotransmitter Systems in Morphologically Undifferentiated Human Y-79 Retinoblastoma Cells: Studies of GABAergic, Glycinergic, and β-Adrenergic Systems

Elements of three neurotransmitter systems were investigated in morphologically undifferentiated human Y-79 retinoblastoma cells in suspension culture. Specific gamma-aminobutyric acid (GABA) uptake, GABA binding, and glycine binding were absent from these cells, although the cells had been shown to exhibit an active uptake and release of [3H]glycine. Binding and competition studies using both alpha- and beta-adrenergic ligands indicated the presence of a beta-adrenergic receptor. This finding was confirmed by treatment of the cells with beta-agonists in competition with a beta-antagonist and with an alpha-antagonist; the level of cyclic AMP was competitively stimulated. Therefore, human Y-79 cells in suspension culture contain beta-adrenergic receptors, and not glycinergic or GABAergic ones. Thus, the Y-79 cells may be of use in studying the factors involved in developmental regulation of neurotransmitter function.     

Study of potent cytotoxic activity of Helleborus cyclophyllus Boiss against a human adenocarcinoma cell line

Helleborus cyclophyllus Boiss is a rhizomatous plant species, with strong allelochemical properties, that has been used since ancient times for its therapeutic properties. In the present study we investigated the ability of an aqueous-soluble fraction of the methanol extract of H. cyclophyllus Boiss leaves, to induce apoptotic cell death on A549 human bronchial epithelial adenocarcinoma cells. A primary human lung fibroblasts’ cell line was used as a model of normal-healthy cells for comparison. Cell morphology was examined after appropriate staining, cytotoxic activity of the extract was determined by the MTT assay, the type of cell death was analyzed by flow cytometry, confirmation of apoptosis was evaluated with the analysis of caspase-3, PARP1 by western blotting, while the chemical composition was assessed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). H. cyclophyllus Boiss extract was selectively active on A549 cells inducing significant morphological changes, even at low concentrations. Characteristic morphological alterations included the release of vesicular formations from A549 cell membranes (ectosomes), detachment of cells from their substrate, generation of a large vesicle into the cytoplasm (thanatosome) and the formation of apoptotic bodies. The selective apoptotic action on treated cells was also confirmed by biochemical criteria. Low concentrations, however, did not affect normal cells. The phytochemical analysis of the extract revealed the presence of cardiac glucosides, bufadienolides and phytoecdysteroids. To the best of our knowledge, the above-mentioned sequences of events leading selectively cancer cells to apoptosis, has not been reported before.Electronic supplementary materialThe online version of this article (10.1007/s10616-020-00425-4) contains supplementary material, which is available to authorized users.     

Size at maturity of Mediterranean marine fishes

In this review we collected data on the length at maturity (L_m) and maximum reported total length (L_max) of 565 Mediterranean marine fish stocks, representing 150 species, 68 families, 24 orders and 3 classes. Overall, L_m ranged from 2 cm, for the males of the toothcarp Aphanius fasciatus, to 350 cm, for the females of the bluntnose sixgill shark Hexanchus griseus. L_m was positively linearly related with L_max for Actinopterygii (logL_m = ?0.123 + 0.92 × logL_max; r ² = 0.87, n = 471, P < 0.001) and Elasmobranchii (logL_m = ?0.008 + 0.922 × logL_max; r ² = 0.90, n = 92, P < 0.001) with the two slopes being significantly different (ANCOVA: F = 2,904, P < 0.001). The reproductive load (L_m/L_max) ranged between 0.23 (sand steenbras Lithognathus mormyrus) and 0.94 (angular roughshark Oxynotus centrina and thornback ray Raja clavata). The mean L_m/L_max was significantly (ANOVA, F = 34.14, P < 0.001) lower for Actinopterygii (mean = 0.59, SD = 0.122, n = 471) compared to Elasmobranchii (mean = 0.70, SD = 0.132, n = 92) and Holocephali (mean = 0.77, SD = 0.077, n = 2). The L_m/L_max was significantly (ANOVA, F = 43.80, P < 0.001) higher for species providing some form of parental care, i.e. guarders, bearers, nesters (mean L_m/L_max ± SD = 0.68 ± 0.141, n = 111) compared to non-guarders (mean L_m/L_max ± SD = 0.59 ± 0.123, n = 454). The mean L_m/L_max displayed a remarkable constancy with longitude (northern and southern Mediterranean coastline: ANOVA, F = 0.01, P = 0.93), latitude (western, central and eastern regions: ANOVA, F = 1.25, P = 0.29) and habitat (ANOVA, F = 0.85, P = 0.51).     

Synergistic Therapeutic Effect of Cisplatin and Phosphatidylinositol 3-Kinase (PI3K) Inhibitors in Cancer Growth and Metastasis of Brca1 Mutant Tumors

Drug resistance and cancer metastasis are two major problems in cancer research. During a course of therapeutic treatment in Brca1-associated tumors, we found that breast cancer stem cells (CSCs) exhibit an intrinsic ability to metastasize and acquire drug resistance through distinct signaling pathways. Microarray analysis indicated that the cytoskeletal remodeling pathway was differentially regulated in CSCs, and this was further evidenced by the inhibitory role of reagents that impair this pathway in the motility of cancer cells. We showed that cisplatin treatment, although initially inhibiting cancer growth, preventing metastasis through blocking cytoskeletal remodeling, and retarding CSC motility, eventually led to drug resistance associated with a marked increase in the number of CSCs. This event was at least partially attributed to the activation of PI3K signaling, and it could be significantly inhibited by co-treatment with rapamycin. These results provide strong evidence that cytoskeletal rearrangement and PI3K/AKT signaling play distinct roles in mediating CSC mobility and viability, respectively, and blocking both pathways synergistically may inhibit primary and metastatic cancer growth.     

Tyrosine Phosphorylation of Wiskott-Aldrich Syndrome Protein (WASP) by Hck Regulates Macrophage Function

We have shown previously that tyrosine phosphorylation of Wiskott-Aldrich syndrome protein (WASP) is important for diverse macrophage functions including phagocytosis, chemotaxis, podosome dynamics, and matrix degradation. However, the specific tyrosine kinase mediating WASP phosphorylation is still unclear. Here, we provide evidence that Hck, which is predominantly expressed in leukocytes, can tyrosine phosphorylate WASP and regulates WASP-mediated macrophage functions. We demonstrate that tyrosine phosphorylation of WASP in response to stimulation with CX3CL1 or via Fcγ receptor ligation were severely reduced in Hck^−/− bone marrow-derived macrophages (BMMs) or in RAW/LR5 macrophages in which Hck expression was silenced using RNA-mediated interference (Hck shRNA). Consistent with reduced WASP tyrosine phosphorylation, phagocytosis, chemotaxis, and matrix degradation are reduced in Hck^−/− BMMs or Hck shRNA cells. In particular, WASP phosphorylation was primarily mediated by the p61 isoform of Hck. Our studies also show that Hck and WASP are required for passage through a dense three-dimensional matrix and transendothelial migration, suggesting that tyrosine phosphorylation of WASP by Hck may play a role in tissue infiltration of macrophages. Consistent with a role for this pathway in invasion, WASP^−/− BMMs do not invade into tumor spheroids with the same efficiency as WT BMMs and cells expressing phospho-deficient WASP have reduced ability to promote carcinoma cell invasion. Altogether, our results indicate that tyrosine phosphorylation of WASP by Hck is required for proper macrophage functions.     

Insights into the role of the (alpha+beta) insertion in the TIM-barrel catalytic domain, regarding the stability and the enzymatic activity of chitinase A from Serratia marcescens

Chitinase A (ChiA) from Serratia marcescens is a mesophilic enzyme with high catalytic activity and high stability. The crystal structure of ChiA has revealed a TIM-barrel fold of the catalytic domain, an (alpha+beta) insertion between the B7 beta-strand and A7 alpha-helix of the TIM-barrel, an FnIII domain at the N-terminus of the molecule and a hinge region that connects the latter to the catalytic domain. In this study, the role of the (alpha+beta) domain on the stability, catalytic activity and specificity of the enzyme was investigated by deleting this domain and studying the enzymatic and structural properties of the resulting truncated enzyme. The obtained data clearly show that by removing the (alpha+beta) domain, the thermal stability of the enzyme is substantially reduced, with an apparent T(m) of 42.0+/-1.0 degrees C, compared to the apparent T(m) of 58.1+/-1.0 degrees C of ChiA at pH 9.0. The specific activity of ChiADelta(alpha+beta) was substantially decreased, the pH optimum was shifted from 6.5 to 5.0 and the substrate and product specificities were altered.     

The mode of lymphoblastoid cell death in response to gas phase cigarette smoke is dose-dependent

Background

Human embryonic stem cells (hESC) have the capacity to differentiate in vivo and in vitro into cells from all three germ lineages. The aim of the present study was to investigate the effect of specific culture conditions on the differentiation of hESC into lung epithelial cells.

Methods

Undifferentiated hESC, grown on a porous membrane in hESC medium for four days, were switched to a differentiation medium for four days; this was followed by culture in air-liquid interface conditions during another 20 days. Expression of several lung markers was measured by immunohistochemistry and by quantitative real-time RT-PCR at four different time points throughout the differentiation and compared to appropriate controls.

Results

Expression of CC16 and NKX2.1 showed a 1,000- and 10,000- fold increase at day 10 of differentiation. Other lung markers such as SP-C and Aquaporin 5 had the highest expression after twenty days of culture, as well as two markers for ciliated cells, FOXJ1 and β-tubulin IV. The results from qRT-PCR were confirmed by immunohistochemistry on paraffin-embedded samples. Antibodies against CC16, SP-A and SP-C were chosen as specific markers for Clara Cells and alveolar type II cells. The functionality was tested by measuring the secretion of CC16 in the medium using an enzyme immunoassay.

Conclusion

These results suggest that by using our novel culture protocol hESC can be differentiated into the major cell types of lung epithelial tissue.     

An experimental and computational investigation of spontaneous lasso formation in microcin J25

The antimicrobial peptide microcin J25 (MccJ25) is posttranslationally matured from a linear preprotein into its native lasso conformation by two enzymes. One of these enzymes cleaves the preprotein and the second enzyme installs the requisite isopeptide bond to establish the lasso structure. Analysis of a mimic of MccJ25 that can be cyclized without the influence of the maturation enzymes suggests that MccJ25 does not spontaneously adopt a near-lasso structure. In addition, we conducted atomistically detailed replica-exchange molecular dynamics simulations of pro-microcin J25 (pro-MccJ25), the 21-residue uncyclized analog of MccJ25, to determine the conformational ensemble explored in the absence of the leader sequence or maturation enzymes. We applied a nonlinear dimensionality reduction technique known as the diffusion map to the simulation trajectories to extract two global order parameters describing the fundamental dynamical motions of the system, and identify three distinct pathways. One path corresponds to the spontaneous adoption of a left-handed lasso, in which the N-terminus wraps around the C-terminus in the opposite sense to the right-handed topology of native MccJ25. Our computational and experimental results suggest a role for the MccJ25 leader sequence and/or its maturation enzymes in facilitating the adoption of the right-handed topology.     

Insulin secretion dynamics of free and alginate-encapsulated insulinoma cells

This study investigates the effect of alginate/poly-l-lysine/alginate (APA) encapsulation on the insulin secretion dynamics exhibited by an encapsulated cell system. Experiments were performed with the aid of a home-built perfusion apparatus providing a 1 min temporal resolution. Insulin profiles were measured from: (i) murine insulinoma βTC3 cells encapsulated in calcium alginate/poly-l-lysine/alginate (APA) beads generated with high guluronic (G) or high mannuoric (M) content alginate, and (ii) murine insulinoma βTC-tet cells encapsulated in high M APA beads and propagated in the presence and absence of tetracycline. Results show that encapsulation in APA beads did not affect the insulin secretion profile shortly post-encapsulation. However, remodeling of the beads due to cell proliferation affected the insulin secretion profiles; and inhibiting remodeling by suppressing cell growth preserved the secretion profile. The implications of these findings regarding the in vivo function of encapsulated insulin secreting cells are discussed.