Zinc does not inhibit prolactin secretion during insulin-induced hypoglycemia in normal men

Zinc (Zn ++) has been shown as an important physiological inhibitor of pituitary PRL release, and Zn ++ and PRL could be involved in a negative feedback regulatory loop. However, this inhibitory effect has not been detected in humans with regard to thyrotropin releasing hormone (TRH), dopamine (DA) and histamine (HA) neurotransmitters. In order to investigate this topic, Zn ++ was acutely and chronically administered to five healthy men to observe the probable inhibitory effect on PRL release during insulin-induced hypoglycemia. The positive PRL response to hypoglycemia has generally been considered to be mediated via the hypothalamus by adrenergic, serotoninergic, histaminergic, opioid-peptidergic and TRH neurotransmitters. The results showed that Zn ++ was not able to inhibit the PRL release during insulin-induced hypoglycemia. Under these conditions, Zn ++ does not block hypothalamic neurotransmitters stimulated by hypoglycemia, thus excluding its clinical application in human beings. On the other hand, the effect of acute stress, such as hypoglycemia, on the serum Zn ++ profile was not observed. ©2006 Dustri-Verlag Dr. K. Feistle.

Chloroquine inhibits Paracoccidioides brasiliensis survival within human monocytes by limiting the availability of intracellular iron

The mechanisms used by Paracoccidioides brasiliensis (Pb 18) to survive into monocytes are not clear. Cellular iron metabolism is of critical importance to the growth of several intracellular pathogens, including P. brasiliensis, whose capacity to multiply in mononuclear phagocytes is dependent on the availability of intracellular iron. Chloroquine, by virtue of its basic properties, has been shown to prevent release of iron from holotransferrin by raising endocytic and lysosomal pH, and thereby interfering with normal iron metabolism. Then, in view of this, we have studied the effects of CHLOR on P. brasiliensis multiplication in human monocytes and its effect on the murine paracoccidioidomycosis. CHLOR induced human monocytes to kill P. brasiliensis. The effect of CHLOR was reversed by FeNTA, an iron compound that is soluble at neutral to alkaline pH, but not by holotransferrin, which releases iron only in an acidic environment. CHLOR treatment of Pb 18-infected BALB/c mice significantly reduced the viable fungi recovery from lungs, during three different periods of evaluation, in a dose-dependent manner. This study demonstrates that iron is of critical importance to the survival of P. brasiliensis yeasts within human monocytes and the CHLOR treatment in vitro induces Pb 18 yeast-killing by monocytes by restricting the availability of intracellular iron. Besides, the CHLOR treatment in vivo significantly reduces the number of organisms in the lungs of Pb-infected mice protecting them from several infections. Thus, CHLOR was effective in the treatment of murine paracoccidioidomycosis, suggesting the potential use of this drug in patients' treatment.

Lack of DNA damage induced by fluoride on mouse lymphoma and human fibroblast cells by single cell gel (comet) assay

Fluoride has widely been used in Dentistry because it is a specific and effective caries prophylactic agent. However, excess fluoride may represent a hazard to human health, especially by causing injury on genetic apparatus. Genotoxicity tests constitute an important part of cancer research for risk assessment of potential carcinogens. In this study, the potential DNA damage associated with exposure to fluoride was assessed by the single cell gel (comet) assay in vitro. Mouse lymphoma and human fibroblast cells were exposed to sodium fluoride (NaF) at final concentration ranging from 7 to 100 μg/mL for 3 h at 37μC. The results pointed out that NaF in all tested concentrations did not contribute to DNA damage as depicted by the mean tail moment and tail intensity for both cellular types assessed. These findings are clinically important because they represent a valuable contribution for evaluation of the potential health risk associated with exposure to agents usually used in dental practice.

Pro- and anti-inflammatory cytokines produced by human monocytes challenged in vitro with Paracoccidioides brasiliensis

Monocytes and macrophages play a central role in innate and adaptive immune response against systemic fungal infections. Imbalances in suppressor or stimulatory cytokine secretion caused by these cells may influence disease development, microorganism death, and the nature of the adaptive immune response. This study analyzed the monocyte cytokine profiles of healthy individuals challenged with high and low virulent strains of P. brasiliensis and mRNA cytokine expression kinetics by reverse transcription polymerase chain reaction (RT-PCR). Peripheral blood monocytes from healthy volunteers were cultured in vitro with and without virulent (Pb18) or low virulence (Pb265) strains from P. brasiliensis viable yeast cells. Interleukin-1 beta (IL-1β), IL-6, IL-8, IL-10, tumor necrosis factor-alpha (TNF-α), and transforming growth factor-beta (TGF-β1) were measured in culture supernatants by enzyme immunoassay (ELISA), and mRNA cytokine expression was determined by RT-PCR at 0, 4, 8, 12, 18 and 48 hr. Both P. brasiliensis strains induced monocyte production of IL-1β, IL-6, IL-10 and TNF-α. Pb18 induced higher levels of IL-1β, IL-6, and IL-10 than Pb265. IL-8 and TGF-β1 levels were not significantly different from those cultured without stimulus. The mRNA cytokine expression was similar to supernatant cytokines measured by ELISA. In vitro monocyte challenge with virulent P. brasiliensis strain induces earlier and higher levels of pro- and anti-inflammatory cytokines than low virulence strain.

Influence of endogenous and synthetic female sex hormones on human blood cells in vitro studied with comet assay

The comet assay has been conducted with numerous cell lines to assess in vitro genotoxicity. In order to use the comet assay as part of an in vitro test for evaluating genotoxicity, however, there are cell-specific factors that need to be better understood. In this present study we have evaluated some factors that may impact upon the DNA damage detected in whole blood (WB) cells and lymphocytes (ILs). Experiments were conducted comparing responses of both cells, and investigating the effects of the female hormonal cycle, and oral contraceptive (OC) use on DNA damage detection in the in vitro comet assay, at three sampling time. No significant differences were detected in the basal levels of DNA damage detected in ILs and WB cells from women OC users and non-users and from men. Basal DNA damage in ILs was unaffected by gender and stage of the menstrual cycle or the stage of the treatment schedule. Our results also indicated that the H2O2 induces DNA damage in human lymphocytes independently of gender, low-dose OC use and hormonal fluctuation. However, data showed that in 3rd sampling of menstrual cycle, lymphocytes were more resistant to H2O2-induced DNA damage than those from OC users and men. © 2007 Elsevier Ltd. All rights reserved.

Inhibition of human neutrophil apoptosis by Paracoccidioides brasiliensis: Role of interleukin-8

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidiodes brasiliensis that presents a wide spectrum of clinical manifestations. Because of the great number of neutrophils polymorphonuclear neutrophils (PMN) found in the P. brasiliensis granuloma, studies have been done to evaluate the role of these cells during the development of the infection. This fungus is found intracellularly in PMN and monocytes/macrophages, suggesting that it is capable of evading damage and surviving inside these cells. Thus, in the present study, we investigated whether P. brasiliensis can prolong the lifetime of PMN, and if this process would be related with IL-8 levels. PMN apoptosis and intracellular levels of IL-8 were analysed by flow cytometry and culture supernatants IL-8 levels were evaluated by enzyme-linked immunosorbent assay. We found that coincubation with P. brasiliensis yeast cells results in an inhibition of PMN apoptosis, which was associated with increase in IL-8 production by these cells. Cocultures treatment with monoclonal antibody anti-IL-8 reversed the inhibitory effect of P. brasiliensis on PMN apoptosis, besides to increase spontaneous apoptosis of these cells. These data show that, in contrast to other microbial pathogens that drive phagocytes into apoptosis to escape killing, P. brasiliensis can extend the lifetime of normal human PMN by inducing autocrine IL-8 production. © 2008 The Authors.

Genotoxicological assessment of two reactive dyes extracted from cotton fibres using artificial sweat

Human eyes have a remarkable ability to recognize hundreds of colour shades, which has stimulated the use of colorants, especially for clothing, but toxicological studies have shown that some textile dyes can be hazardous to human health. Under conditions of intense perspiration, dyes can migrate from coloured clothes and penetrate into human skin. Garments made from cotton fabrics are the most common clothing in tropical countries, due to their high temperatures. Aiming to identify safe textile dyes for dyeing cotton fabrics, the genotoxicity [in vitro Comet assay with normal human dermal fibroblasts (NHDF), Tail Intensity] and mutagenicity [Salmonella/microsome preincubation assay (30 min), tester strains TA98, TA100, YG1041 and YG1042] of Reactive Blue 2 (RB2, CAS No. 12236-82-7, C.I. 61211) and Reactive Green 19 (RG19, CAS No. 61931-49-5, C.I. 205075) were evaluated both in the formulated form and as extracted from cotton fibres using different artificial sweats. Both the dyes could migrate from cotton fibres to sweat solutions, the sweat composition and pH being important factors during this extraction. However, the dye sweat solutions showed no genotoxic/mutagenic effects, whereas a weak mutagenic potential was detected by the Ames test for both dyes in their formulated form. These findings emphasize the relevance of textile dyes assessment under conditions that more closely resemble human exposure, in order to recognize any hazard. © 2013 Elsevier Ltd. All rights reserved.

Finasteride Inhibits Human Prostate Cancer Cell Invasion through MMP2 and MMP9 Downregulation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Is it safe to utilize in vitro reconstituted human oral epithelium? An oncogenetic pathway study

Cell therapy is a therapeutic strategy used to replace or repair damaged tissue. The epithelium transplantation of cultivated keratinocytes has been applied to several modalities of reconstruction, like oral, urethra and ocular surface. Life and death signals work coordinately to ensure cellular quality control and the viability of an organism. The aim of this study is to verify that culture conditions did not induce genetic mutations through the analysis of the key genes: pAKT, Pten, p53 and MDM2 and investigate the presence of the related proteins in human oral keratinocytes obtained by primary culture and in vitro cultivated. Formalin fixed and paraffin embedded tissues from the oral cavity were utilized as control for normal expression of the related markers and two oral squamous cell carcinoma cell lines provided the expression pattern of the proposed markers in the event of cellular transformation. Akt, PTEN, p53 and MDM2 immunohistochemistry and Western-Blotting analyzes were performed. The results showed the expression levels and intracellular localizations of the four proteins evaluated. These analyzes confirmed that the produced in vitro epithelium is bio-compatible for its utilization as reconstruction and reparatory tissue, however further analyses and additional research on other biomarkers should be performed to analyse the long term engraftment of transplantable primary culture of oral keratinocytes and the long term resistance to cellular transformation.

Untersuchungen zur Beeinflussung der Reparatur oxidativer DNA-Schäden durch Poly(ADP-Ribose)-Polymerase, AP-Endonuklease 1 und das Xeroderma pigmentosum A Protein

Gegenstand dieser Arbeit war die Untersuchung der Bedeutung der Poly(ADP-Ribose)-Polymerase 1 (PARP 1), der AP Endonuklease 1 (Ape 1) und des Xeroderma pigmentosum A (XPA) Proteins für die DNA-Reparatur in Säugerzellen.Zunächst wurde der Einfluss der PARP 1-Aktivität auf die Reparatur verschiedener DNA-Modifikationen untersucht. Die Ergebnisse zeigen erstmalig, dass eine Hemmung der PARP-Aktivität nicht nur eine deutliche Verlangsamung der Reparatur von Einzelstrangbrüchen, sondern auch von oxidativen Purinmodifikationen und Pyrimidindimeren zur Folge hat. Interessanterweise erfolgte diese Verlangsamung der DNA-Reparatur nicht in Csb-defizienten Zellen. Diese Ergebnisse deuten darauf hin, dass die Aktivierung der PARP 1 und das Csb-Protein zusammen an einem neuartigen Mechanismus beteiligt sind, der die globale Reparatur verschiedener DNA-Modifikationen beschleunigt.Weiterhin wurde die Bedeutung der Nukleotidexcisionsreparatur als back-up Reparatur von 8 Hydroxyguanin untersucht. Dazu wurden normale und XPA-defiziente Fibroblasten des Menschen mit einem hOgg1-anitsense Konstrukt transfiziert und dann in diesen Zellen die Reparaturkinetiken oxidativer Basenmodifikationen bestimmt. Dadurch konnte eine Beteiligung des XPA-Proteins an diesem Reparaturweg ausgeschlossen werden.Außerdem wurden die Auswirkungen einer AP Endonuklease-1-Überexpression in XRCC1-defizienten Zellen auf die Reparatur von Einzelstrangbrüchen untersucht. Die Reparatur der induzierten Einzelstrangbrüche war in XRCC1-defizienten Zellen erwartungsgemäß deutlich langsamer als in XRCC1-profizienten Zellen. Die Überexpression der AP Endonuklease 1 in XRCC1-defizienten Zellen führte zu einer teilweisen Beschleunigung der Einzelstrangbruchreparatur.

New computational biology tools for the systematic analysis of the structure and expression of human genes

From the late 1980s, the automation of sequencing techniques and the computer spread gave rise to a flourishing number of new molecular structures and sequences and to proliferation of new databases in which to store them. Here are presented three computational approaches able to analyse the massive amount of publicly avalilable data in order to answer to important biological questions. The first strategy studies the incorrect assignment of the first AUG codon in a messenger RNA (mRNA), due to the incomplete determination of its 5' end sequence. An extension of the mRNA 5' coding region was identified in 477 in human loci, out of all human known mRNAs analysed, using an automated expressed sequence tag (EST)-based approach. Proof-of-concept confirmation was obtained by in vitro cloning and sequencing for GNB2L1, QARS and TDP2 and the consequences for the functional studies are discussed. The second approach analyses the codon bias, the phenomenon in which distinct synonymous codons are used with different frequencies, and, following integration with a gene expression profile, estimates the total number of codons present across all the expressed mRNAs (named here "codonome value") in a given biological condition. Systematic analyses across different pathological and normal human tissues and multiple species shows a surprisingly tight correlation between the codon bias and the codonome bias. The third approach is useful to studies the expression of human autism spectrum disorder (ASD) implicated genes. ASD implicated genes sharing microRNA response elements (MREs) for the same microRNA are co-expressed in brain samples from healthy and ASD affected individuals. The different expression of a recently identified long non coding RNA which have four MREs for the same microRNA could disrupt the equilibrium in this network, but further analyses and experiments are needed.

High expression of NPY receptors in the human testis

NPY receptors represent novel molecular therapeutic targets in cancer and obesity. However, the extent of NPY receptor expression in normal human tissues is poorly investigated. Based on the role of NPY in reproductive functions, the NPY receptor expression was studied in 25 normal human testes and, additionally, 24 testicular tumors using NPY receptor autoradiography. In the normal testis, Leydig cells strongly expressed NPY receptor subtype Y2, and small arterial blood vessels Y1. Y2 receptors were found to be functional with agonist-stimulated [(35)S]GTPγS binding autoradiography. Full functional integrity of the NPY system was further suggested by the immunohistochemical detection of NPY peptide in nerve fibers directly adjacent to Leydig cells and arteries. Germ cell tumors expressed Y1 and Y2 on tumor cells in 33% and Y1 on intratumoral blood vessels in 50%. Based on its strong NPY receptor expression in Leydig cells and blood vessels, the normal human testis represents a potentially important physiological and pharmalogical NPY target.

Interferon-inducible expression of APOBEC3 editing enzymes in human hepatocytes and inhibition of hepatitis B virus replication

Hypermutations in hepatitis B virus (HBV) DNA by APOBEC3 cytidine deaminases have been detected in vitro and in vivo, and APOBEC3G (A3G) and APOBEC3F (A3F) have been shown to inhibit the replication of HBV in vitro, but the presumably low or even absent hepatic expression of these enzymes has raised the question as to their physiological impact on HBV replication. We show that normal human liver expresses the mRNAs of APOBEC3B (A3B), APOBEC3C (A3C), A3F, and A3G. In primary human hepatocytes, interferon alpha (IFN-alpha) stimulated the expression of these cytidine deaminases up to 14-fold, and the mRNAs of A3G, A3F, and A3B reached expression levels of 10%, 3%, and 3%, respectively, relative to GAPDH mRNA abundance. On transfection, the full-length protein A3B(L) inhibited HBV replication in vitro as efficiently as A3G or A3F, whereas the truncated splice variant A3B(S) and A3C had no effect. A3B(L) and A3B(S) were detected predominantly in the nucleus of uninfected cells; however, in HBV-expressing cells both proteins were found also in the cytoplasm and were associated with HBV viral particles, similarly to A3G and A3F. Moreover, A3G, A3F, and A3B(L), but not A3B(S), induced extensive G-to-A hypermutations in a fraction of the replicated HBV genomes. In conclusion, the editing enzymes A3B(L), A3F, and most markedly A3G, which are expressed in liver and up-regulated by IFN-alpha in hepatocytes, are candidates to contribute to the noncytolytic clearance of HBV.

Human microbiota-secreted factors inhibit shiga toxin synthesis by enterohemorrhagic Escherichia coli O157:H7

Escherichia coli O157:H7 is a food-borne pathogen causing hemorrhagic colitis and hemolytic-uremic syndrome, especially in children. The main virulence factor responsible for the more serious disease is the Shiga toxin 2 (Stx2), which is released in the gut after oral ingestion of the organism. Although it is accepted that the amount of Stx2 produced by E. coli O157:H7 in the gut is critical for the development of disease, the eukaryotic or prokaryotic gut factors that modulate Stx2 synthesis are largely unknown. In this study, we examined the influence of prokaryotic molecules released by a complex human microbiota on Stx2 synthesis by E. coli O157:H7. Stx2 synthesis was assessed after growth of E. coli O157:H7 in cecal contents of gnotobiotic rats colonized with human microbiota or in conditioned medium having supported the growth of complex human microbiota. Extracellular prokaryotic molecules produced by the commensal microbiota repress stx(2) mRNA expression and Stx2 production by inhibiting the spontaneous and induced lytic cycle mediated by RecA. These molecules, with a molecular mass of below 3 kDa, are produced in part by Bacteroides thetaiotaomicron, a predominant species of the normal human intestinal microbiota. The microbiota-induced stx(2) repression is independent of the known quorum-sensing pathways described in E. coli O157:H7 involving SdiA, QseA, QseC, or autoinducer 3. Our findings demonstrate for the first time the regulatory activity of a soluble factor produced by the complex human digestive microbiota on a bacterial virulence factor in a physiologically relevant context.

MicroRNA-140 is expressed in differentiated human articular chondrocytes and modulates interleukin-1 responses

OBJECTIVE: MicroRNA (miRNA) are a class of noncoding small RNAs that act as negative regulators of gene expression. MiRNA exhibit tissue-specific expression patterns, and changes in their expression may contribute to pathogenesis. The objectives of this study were to identify miRNA expressed in articular chondrocytes, to determine changes in osteoarthritic (OA) cartilage, and to address the function of miRNA-140 (miR-140). METHODS: To identify miRNA specifically expressed in chondrocytes, we performed gene expression profiling using miRNA microarrays and quantitative polymerase chain reaction with human articular chondrocytes compared with human mesenchymal stem cells (MSCs). The expression pattern of miR-140 was monitored during chondrogenic differentiation of human MSCs in pellet cultures and in human articular cartilage from normal and OA knee joints. We tested the effects of interleukin-1beta (IL-1beta) on miR-140 expression. Double-stranded miR-140 (ds-miR-140) was transfected into chondrocytes to analyze changes in the expression of genes associated with OA. RESULTS: Microarray analysis showed that miR-140 had the largest difference in expression between chondrocytes and MSCs. During chondrogenesis, miR-140 expression in MSC cultures increased in parallel with the expression of SOX9 and COL2A1. Normal human articular cartilage expressed miR-140, and this expression was significantly reduced in OA tissue. In vitro treatment of chondrocytes with IL-1beta suppressed miR-140 expression. Transfection of chondrocytes with ds-miR-140 down-regulated IL-1beta-induced ADAMTS5 expression and rescued the IL-1beta-dependent repression of AGGRECAN gene expression. CONCLUSION: This study shows that miR-140 has a chondrocyte differentiation-related expression pattern. The reduction in miR-140 expression in OA cartilage and in response to IL-1beta may contribute to the abnormal gene expression pattern characteristic of OA.