Sources and fates of perchlorate in soils in Chile: A case study of perchlorate dynamics in soil-crop systems using lettuce (Lactuca sativa) fields.

Perchlorate occurs naturally in the environment in deposits of nitrate and can be formed in the atmosphere and precipitate into soil. However, little is known about the occurrence and levels of perchlorate in soils and fertilizers in Chile and its impacts on agricultural systems and food safety. In this study, concentrations of perchlorate were determined in 101 surface soils and 17 fertilizers [nitrogenous (n = 8), nitrogen-phosphorous-potassium (NPK; n = 3), phosphate (n = 2) and non-nitrogenous (n = 4)] collected across Chile from 2017 to 2018. Our results show that perchlorate was detected mainly in agricultural soils (mean: 0.32 ng g-1), grassland rotation sites (0.41 ngg-1) and urban locations (0.38 ng g-1). Interestingly, elevated concentrations of perchlorate (9.66 and 54.0 ng g-1) were found in agricultural soils. All fertilizers contained perchlorate: nitrogenous fertilizers (mean: 32.6 mg kg-1), NPK (mean: 12.6 mg kg-1), non-nitrogenous fertilizers (mean: 10.2 mg kg-1) and phosphates (mean: 11.5 mg kg-1). Only one type of nitrogenous fertilizer (KNO3: 95.3 mg kg-1) exceeded the international regulation limit (50 mg kg-1). For two agronomic practices, the content of perchlorate in lettuce increased as the fertilizer application rate increased, with fertigation promoting a more significant accumulation. However, the concentrations generally remained below regulatory values. Our results suggest that fertilizers constitute an important source of perchlorate in soils.

Preclinical study for treatment of hypospadias by advanced therapy medicinal products.

PURPOSE: This paper explores the feasibility of a new therapy for the treatment of hypospadias patients. Hypospadias is a very common congenital malformation of male genitals, with very high rate of recurrences after surgery. The field of regenerative medicine, which offers innovative solutions for many pathologies, still does not offer reliable solution for this pathology. Here, we propose quality, safety, and clinical feasibility assessment for an oral mucosa advanced therapy medicinal product (ATMP) grown on a biocompatible scaffold for a clinical study on urethral reconstruction of hypospadias patients. METHODS: Urethral and oral mucosal epithelia from donor biopsies were cultivated between two fibrin layers, under clinical-grade conditions for cell and tissue characterization and comparison, aimed at tissue engineering. In addition, single-clone analyses were performed to analyze gene expression profiles of the two epithelia by microarray technology. RESULTS: Oral mucosa appeared suitable for urethral reconstruction. The resulting ATMP was proven to maintain stem cells and regenerative potency. The preclinical safety studies were performed on human tissues to assess abnormalities and tumorigenicity, and confirmed the safety of the ATMP. Finally, the patient selection and the clinical protocol for the upcoming clinical trial were defined. CONCLUSIONS: Against this backdrop, in this paper, we are proposing a new reproducible and reliable ATMP for the treatment of hypospadias.

Expression of p89(c-Mybex9b), an alternatively spliced form of c-Myb, is required for proliferation and survival of p210BCR/ABL-expressing cells.

The c-Myb gene encodes the p75(c-Myb) isoform and less-abundant proteins generated by alternatively spliced transcripts. Among these, the best known is p(c-Mybex9b), which contains 121 additional amino acids between exon 9 and 10, in a domain involved in protein-protein interactions and negative regulation. In hematopoietic cells, expression of p(c-Mybex9b) accounts for 10-15% of total c-Myb; these levels may be biologically relevant because modest changes in c-Myb expression affects proliferation and survival of leukemic cells and lineage choice and frequency of normal hematopoietic progenitors. In this study, we assessed biochemical activities of p(c-Mybex9b) and the consequences of perturbing its expression in K562 and primary chronic myeloid leukemia (CML) progenitor cells. Compared with p75(c-Myb), p(c-Mybex9b) is more stable and more effective in transactivating Myb-regulated promoters. Ectopic expression of p(c-Mybex9b) enhanced proliferation and colony formation and reduced imatinib (IM) sensitivity of K562 cells; conversely, specific downregulation of p(c-Mybex9b) reduced proliferation and colony formation, enhanced IM sensitivity of K562 cells and markedly suppressed colony formation of CML CD34(+) cells, without affecting the levels of p75(c-Myb). Together, these studies indicate that expression of the low-abundance p(c-Mybex9b) isoform has an important role for the overall biological effects of c-Myb in BCR/ABL-transformed cells.

An intronic mutation in MLH1 associated with familial colon and breast cancer.

Single base substitutions can lead to missense mutations, silent mutations or intronic mutations, whose significance is uncertain. Aberrant splicing can occur due to mutations that disrupt or create canonical splice sites or splicing regulatory sequences. The assessment of their pathogenic role may be difficult, and is further complicated by the phenomenon of alternative splicing. We describe an HNPCC patient, with early-onset colorectal cancer and a strong family history of colorectal and breast tumors, who harbours a germ line MLH1 intronic variant (IVS9 c.790 +4A>T). The proband, together with 2 relatives affected by colorectal-cancer and 1 by breast cancer, have been investigated for microsatellite instability, immunohistochemical MMR protein staining, direct sequencing and Multiplex Ligation-dependent Probe Amplification. The effect of the intronic variant was analyzed both by splicing prediction software and by hybrid minigene splicing assay. In this family, we found a novel MLH1 germline intronic variant (IVS9 c.790 +4A>T) in intron 9, consisting of an A to T transversion, in position +4 of the splice donor site of MLH1. The mutation is associated with the lack of expression of the MLH1 protein and MSI in tumour tissues. Furthermore, our results suggest that this substitution leads to a complete skip of both exon 9 and 10 of the mutant allele. Our findings suggest that this intronic variant plays a pathogenic role.

Coding sequence and intron-exon junctions of the c-myb gene are intact in the chronic phase and blast crisis stages of chronic myeloid leukemia patients.

The c-myb gene encodes a transcription factor required for proliferation, differentiation and survival of normal and leukemic hematopoietic cells. c-Myb has a longer half-life in BCR/ABL-expressing than in normal cells, a feature which depends, in part, on PI-3K/Akt-dependent regulation of proteins interacting with the leucine zipper/negative regulatory region of c-Myb. Thus, we asked whether the stability of c-Myb in leukemic cells might be enhanced by mutations interfering with its degradation. We analyzed the c-myb gene in 133 chronic myeloid leukemia (CML) patients in chronic phase and/or blast crisis by denaturing-high performance liquid chromatography (D-HPLC) and sequence analysis of PCR products corresponding to the entire coding sequence and each exon-intron boundary. No mutations were found. We found four single nucleotide polymorphisms (SNPs) and identified an alternatively spliced transcript lacking exon 5, but SNPs frequency and expression of the alternatively spliced transcript were identical in normal and CML cells. Thus, the enhanced stability of c-Myb in CML blast crisis cells and perhaps in other types of leukemia is not caused by a genetic mechanism.

Expression of CCL9/MIP-1gamma is repressed by BCR/ABL and its restoration suppresses in vivo leukemogenesis of 32D-BCR/ABL cells.

Transformation of hematopoietic cells by the BCR/ABL oncogene is caused by perturbation of signal transduction pathways leading to altered patterns of gene expression and activity. By oligonucleotide microarray hybridization of polysomal RNA of untreated and STI571-treated 32D-BCR/ABL cells, we identified the beta-chemokine CCL9 as a gene regulated by BCR/ABL in a tyrosine kinase-dependent manner. BCR/ABL repressed CCL9 expression at the transcriptional level by mechanisms involving suppression of p38 MAP kinase, and modulation of the activity of CDP/cut and C/EBPalpha, two transcription regulators of myeloid differentiation. However, repression of C/EBP-dependent transcription did not prevent the induction of CCL9 expression by STI571, suggesting that C/EBPalpha is involved in maintaining rather than in inducing CCL9 expression. Restoration of CCL9 expression in 32D-BCR/ABL cells had no effect on the in vitro proliferation of these cells, but reduced their leukemogenic potential in vivo, possibly by recruitment of CD3-positive immune cells. Together, these findings suggest that downregulation of chemokine expression may be involved in BCR/ABL-dependent leukemogenesis by altering the relationship between transformed cells and the microenvironment.

Formyl-methionyl-leucyl-phenylalanine induces prostaglandin E2 release from human amnion-derived WISH cells by phospholipase C-mediated [Ca+]i rise.

The presence of binding sites for formyl-methionyl-leucyl-phenylalanine (fMLP), its effect on prostaglandin E (PGE) release, and the signal transduction pathway activated by the peptide were investigated in human amnion-derived WISH cells. Our results demonstrate that specific binding sites for fMLP are present on WISH cells and that the peptide induces a significant increase of prostaglandin (PG)E2 release. The kinetic properties of binding are similar to those previously found in amnion tissue prior to the onset of labor, i.e., only one population of binding sites with low affinity for the peptide is present. Binding of 3H-fMLP in WISH cells is inhibited by N-t-butoxycarbonyl-methionyl-leucyl-phenylalanine, an fMLP receptor antagonist, with an IC50 value very close to that shown by nonlaboring amnion. The fMLP-induced PGE2 output is inhibited by indomethacin, quinacrine, and U-73122, inhibitors of cyclooxygenase, phospholipase A2, and phospholipase C, respectively. As regards the transduction pathway activated by fMLP, we demonstrate that phospholipase C activation, followed by an increase of intracellular calcium concentration ([Ca2+]i), is involved in response to the peptide. Our results add further evidence to the role of proinflammatory agents in the determination of labor. Furthermore, because WISH cells appear to behave like nonlaboring amnion tissue, they represent the ideal candidate for in vitro investigation of the events triggering the mechanism of delivery.

Influence of oxytocin on prostaglandin E2, intracellular calcium, and cyclic adenosine monophosphate in human amnion-derived (WISH) cells.

OBJECTIVE: This study was undertaken to investigate the regulation of prostaglandin release by oxytocin and the influence of oxytocin on intracellular calcium and on the cyclic adenosine monophosphate system in human amnion-derived WISH cells. STUDY DESIGN: We determined prostaglandin E(2) release from WISH cells treated with oxytocin, evaluated the cytosolic calcium concentration in single WISH cells by confocal microscopy, and measured both intracellular cyclic adenosine monophosphate levels and adenylyl cyclase activity after oxytocin treatment. RESULTS: Treatment of WISH cells with oxytocin resulted in a concentration-dependent release of prostaglandin E(2), which was increased by lithium chloride and inhibited by indomethacin and U-73122. In single WISH cells, oxytocin increased cytosolic calcium. Moreover, the hormone lowered levels of intracellular cyclic adenosine monophosphate but did not alter adenylyl cyclase activity. CONCLUSIONS: Our data demonstrate, for the first time, that WISH cells respond to oxytocin by increasing prostaglandin E(2) release. In addition to phospholipase C activation and cytosolic calcium increase, the hormone effect involves also a reduction of the cyclic adenosine monophosphate level.

The DNA-binding drugs mithramycin and chromomycin are powerful inducers of erythroid differentiation of human K562 cells.

The human leukaemic K562 cell line can be induced in vitro to undergo erythroid differentiation by a variety of chemical compounds, including haemin, butyric acid, 5-azacytidine and cytosine arabinoside. Differentiation of K562 cells is associated with an increased expression of embryo-fetal globin genes, such as the zeta, epsilon and gamma globin genes. Therefore the K562 cell line has been proposed as a useful in vitro model system to determine the therapeutic potential of new differentiating compounds as well as to study the molecular mechanism(s) regulating changes in the expression of embryonic and fetal human globin genes. Inducers of erythroid differentiation which stimulate gamma-globin synthesis could be considered for possible use in the experimental therapy of those haematological diseases associated with a failure in the expression of adult beta-globin genes. In this paper we demonstrated that the G + C selective DNA-binding drugs chromomycin and mithramycin were powerful inducers of erythroid differentiation of K562 cells. Erythroid differentiation was associated with an increase in the accumulation of (a) Hb Gower 1 and Hb Portland and (b) gamma-globin mRNA.

Human leukemia K562 cells: induction to erythroid differentiation by guanine, guanosine and guanine nucleotides.

BACKGROUND AND OBJECTIVE: Human leukemic K562 cells are able to undergo erythroid differentiation in vitro when cultured with a variety of inducers, leading to increased expression of embryo-fetal globin genes such as the zita, epsilon and gamma-globin genes. Therefore the K562 cell line has been proposed as a very useful in vitro model system for determining the therapeutical potential of new differentiating compounds as well as for studying the molecular mechanism(s) that regulate changes in the expression of embryonic and fetal human globin genes. In this study we explored whether nucleoside triphosphates and related compounds are able to induce differentiation of K562 cells. METHODS: K562 cell differentiation was studied using the benzidine test; hemoglobins were characterized by cellulose acetate gel electrophoresis and mRNA accumulation was investigated by Northern blot analysis. RESULTS: The main conclusion of this paper is that guanine, guanosine and guanine ribonucleotides are effective inducers of K562 cell differentiation. Expression of both Hb Portland and Hb Gower 1 is increased in GTP-induced K562 cells. This increase is associated with greater gamma-globin mRNA accumulation. By contrast, ATP, CTP and UTP are not able to induce erythroid differentiation. INTERPRETATION AND CONCLUSIONS: These findings suggest that guanine, guanosine and guanine ribonucleotides are inducers of erythroid differentiation of K562 cells. This is of some relevance since differentiating compounds have been proposed as antitumor agents. In addition, inducers of erythroid differentiation that stimulate gamma-globin synthesis might be considered in the experimental therapy of hematological diseases associated with a failure in the expression of adult beta-globin genes.

Adenosine analogs inhibit acetylcholine release and cyclic AMP synthesis in the guinea-pig superior cervical ganglion.

The ability of adenosine agonists to modulate the electrically evoked release of acetylcholine (ACh) from [3H]choline preloaded guinea-pig superior cervical ganglia (SCG) was investigated. The adenosine A1-receptor selective agonist N6-cyclohexyladenosine (CHA) and 2-chloroadenosine (2-CADO) inhibited the evoked transmitter release, the effect being reversed by the A1-receptor selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and by sulmazole (SUL), which blocks both the A1-receptor and the adenylate cyclase inhibitory regulator Gi. In whole ganglia, CHA decreased both the basal and the forskolin (FSK)-stimulated cyclic AMP synthesis. The latter effect was again prevented by the A1 antagonist DPCPX. These results are compatible with the existence, in the guinea-pig SCG, of adenosine A1-receptors, part of which are located on the presynaptic nerve terminals mediating an inhibition of ACh release.

Analysis of heavy metals in Aceto Balsamico Tradizionale di Modena by flame atomic absorption spectroscopy.

A detailed investigation of Aceto Balsamico Tradizionale di Modena (ABTM) was carried out. The necessity of knowing the metal content of this original and widely used product is of great importance. Measurements were carried out on a large number of samples of different ages to check the dependence of metal content on product aging.