Nanomolar Levels Of Pahs In Extracts From Urban Air Induce Mapk Signaling In Hepg2 Cells.

Polycyclic aromatic hydrocarbons (PAHs) are common environmental pollutants that occur naturally in complex mixtures. Many of the adverse health effects of PAHs including cancer are linked to the activation of intracellular stress response signaling. This study has investigated intracellular MAPK signaling in response to PAHs in extracts from urban air collected in Stockholm, Sweden and Limeira, Brazil, in comparison to BP in HepG2 cells. Nanomolar concentrations of PAHs in the extracts induced activation of MEK4 signaling with down-stream increased gene expression of several important stress response mediators. Involvement of the MEK4/JNK pathway was confirmed using siRNA and an inhibitor of JNK signaling resulting in significantly reduced MAPK signaling transactivated by the AP-1 transcription factors ATF2 and c-Jun. ATF2 was also identified as a sensitive stress responsive protein with activation observed at extract concentrations equivalent to 0.1 nM BP. We show that exposure to low levels of environmental PAH mixtures more strongly activates these signaling pathways compared to BP alone suggesting effects due to interactions. Taken together, this is the first study showing the involvement of MEK4/JNK/AP-1 pathway in regulating the intracellular stress response after exposure to nanomolar levels of PAHs in environmental mixtures.

Treadmill Training Increases Sirt-1 And Pgc-1 α Protein Levels And Ampk Phosphorylation In Quadriceps Of Middle-aged Rats In An Intensity-dependent Manner.

The present study investigated the effects of running at 0.8 or 1.2 km/h on inflammatory proteins (i.e., protein levels of TNF- α , IL-1 β , and NF- κ B) and metabolic proteins (i.e., protein levels of SIRT-1 and PGC-1 α , and AMPK phosphorylation) in quadriceps of rats. Male Wistar rats at 3 (young) and 18 months (middle-aged rats) of age were divided into nonexercised (NE) and exercised at 0.8 or 1.2 km/h. The rats were trained on treadmill, 50 min per day, 5 days per week, during 8 weeks. Forty-eight hours after the last training session, muscles were removed, homogenized, and analyzed using biochemical and western blot techniques. Our results showed that: (a) running at 0.8 km/h decreased the inflammatory proteins and increased the metabolic proteins compared with NE rats; (b) these responses were lower for the inflammatory proteins and higher for the metabolic proteins in young rats compared with middle-aged rats; (c) running at 1.2 km/h decreased the inflammatory proteins and increased the metabolic proteins compared with 0.8 km/h; (d) these responses were similar between young and middle-aged rats when trained at 1.2 km. In summary, the age-related increases in inflammatory proteins, and the age-related declines in metabolic proteins can be reversed and largely improved by treadmill training.

Pluripotent Reprogramming of Fibroblasts by Lentiviralmediated Insertion of SOX2, C-MYC, and TCL-1A

Reprogramming of somatic cells to pluripotency promises to boost cellular therapy. Most instances of direct reprogramming have been achieved by forced expression of defined exogenous factors using multiple viral vectors. The most used 4 transcription factors, octamer-binding transcription factor 4 (OCT4), (sex determining region Y)-box 2 (SOX2), Kruppel-like factor 4 (KLF4), and v-myc myelocytomatosis viral oncogene homolog (C-MYC), can induce pluripotency in mouse and human fibroblasts. Here, we report that forced expression of a new combination of transcription factors (T-cell leukemia/lymphoma protein 1A [TCL-1A], C-MYC, and SOX2) is sufficient to promote the reprogramming of human fibroblasts into pluripotent cells. These 3-factor pluripotent cells are similar to human embryonic stem cells in morphology, in the ability to differentiate into cells of the 3 embryonic layers, and at the level of global gene expression. Induced pluripotent human cells generated by a combination of other factors will be of great help for the understanding of reprogramming pathways. This, in turn, will allow us to better control cell-fate and apply this knowledge to cell therapy.

The CEBPA gene is down-regulated in acute promyelocytic leukemia and its upstream promoter, but not the core promoter, is highly methylated

impairment of CCAAT Enhancer Binding Protein alpha (CEBPA) function is a common finding in acute myeloid leukemia; nevertheless, its relevance for acute promyelocytic leukemia pathogenesis is unclear. We analyzed the expression and assessed the methylation status of the core and upstream promoters of CEBPA in acute promyelocytic leukemia at diagnosis. Patients with acute promyelocytic leukemia (n=18) presented lower levels of CEBPA expression compared to healthy controls (n=5), but higher levels than those in acute myeloid leukemia with t(8;21) (n=9) and with inv(16) (n=5). Regarding the core promoter, we detected no methylation in 39 acute promyelocytic leukemia samples or in 8 samples from controls. In contrast, analysis of the upstream promoter showed methylation in 37 of 39 samples, with 17 patients showing methylation levels over 30%. Our results corroborate data obtained in animal models showing that CEBPA is down-regulated in acute promyelocytic leukemia stem cells and suggest that epigenetic mechanisms may be involved.

Vaccination of Mice with Salmonella Expressing VapA: Mucosal and Systemic Th1 Responses Provide Protection against Rhodococcus equi Infection

Conventional vaccines to prevent the pneumonia caused by Rhodococcus equi have not been successful. We have recently demonstrated that immunization with Salmonella enterica Typhimurium expressing the VapA antigen protects mice against R. equi infection. We now report that oral vaccination of mice with this recombinant strain results in high and persistent fecal levels of antigen-specific IgA, and specific proliferation of the spleen cells of immunized mice in response to the in vitro stimulation with R. equi antigen. After in vitro stimulation, spleen cells of immunized mice produce high levels of Th1 cytokines and show a prominent mRNA expression of the Th1 transcription factor T-bet, in detriment of the Th2 transcription factor GATA-3. Following R. equi challenge, a high H(2)O(2), NO, IL-12, and IFN-gamma content is detected in the organs of immunized mice. On the other hand, TNF-alpha and IL-4 levels are markedly lower in the organs of vaccinated mice, compared with the non-vaccinated ones. The IL-10 content and the mRNA transcription level of TGF-beta are also higher in the organs of immunized mice. A greater incidence of CD4(+) and CD8(+) T cells and B lymphocytes is verified in vaccinated mice. However, there is no difference between vaccinated and non-vaccinated mice in terms of the frequency of CD4(+)CD25(+)Foxp3(+) T cells. Finally, we show that the vaccination confers a long-term protection against R. equi infection. Altogether, these data indicate that the oral vaccination of mice with S. enterica Typhimurium expressing VapA induces specific and long-lasting humoral and cellular responses against the pathogen, which are appropriately regulated and allow tissue integrity after challenge.

Exercise training and caloric restriction prevent reduction in cardiac Ca(2+)-handling protein profile in obese rats

Previous studies show that exercise training and caloric restriction improve cardiac function in obesity. However, the molecular mechanisms underlying this effect on cardiac function remain unknown. Thus, we studied the effect of exercise training and/or caloric restriction on cardiac function and Ca(2+) handling protein expression in obese rats. To accomplish this goal, male rats fed with a high-fat and sucrose diet for 25 weeks were randomly assigned into 4 groups: high-fat and sucrose diet, high-fat and sucrose diet and exercise training, caloric restriction, and exercise training and caloric restriction. An additional lean group was studied. The study was conducted for 10 weeks. Cardiac function was evaluated by echocardiography and Ca(2+) handling protein expression by Western blotting. Our results showed that visceral fat mass, circulating leptin, epinephrine, and norepinephrine levels were higher in rats on the high-fat and sucrose diet compared with the lean rats. Cardiac nitrate levels, reduced/oxidized glutathione, left ventricular fractional shortening, and protein expression of phosphorylated Ser(2808)-ryanodine receptor and Thr(17-)phospholamban were lower in rats on the high-fat and sucrose diet compared with lean rats. Exercise training and/or caloric restriction prevented increases in visceral fat mass, circulating leptin, epinephrine, and norepinephrine levels and prevented reduction in cardiac nitrate levels and reduced: oxidized glutathione ratio. Exercise training and/or caloric restriction prevented reduction in left ventricular fractional shortening and in phosphorylation of the Ser(2808)-ryanodine receptor and Thr(17)-phospholamban. These findings show that exercise training and/or caloric restriction prevent cardiac dysfunction in high-fat and sucrose diet rats, which seems to be attributed to decreased circulating neurohormone levels. In addition, this nonpharmacological paradigm prevents a reduction in the Ser(2808)-ryanodine receptor and Thr(17-)phospholamban phosphorylation and redox status. (Hypertension. 2010;56:629-635.)

In silico analysis of candidate genes involved in light sensing and signal transduction pathways in soybean

Several aspects of photoperception and light signal transduction have been elucidated by studies with model plants. However, the information available for economically important crops, such as Fabaceae species, is scarce. In order to incorporate the existing genomic tools into a strategy to advance soybean research, we have investigated publicly available expressed sequence tag ( EST) sequence databases in order to identify Glycine max sequences related to genes involved in light-regulated developmental control in model plants. Approximately 38,000 sequences from open-access databases were investigated, and all bona fide and putative photoreceptor gene families were found in soybean sequence databases. We have identified G. max orthologs for several families of transcriptional regulators and cytoplasmic proteins mediating photoreceptor-induced responses, although some important Arabidopsis phytochrome-signaling components are absent. Moreover, soybean and Arabidopsis gene-family homologs appear to have undergone a distinct expansion process in some cases. We propose a working model of light perception, signal transduction and response-eliciting in G. max, based on the identified key components from Arabidopsis. These results demonstrate the power of comparative genomics between model systems and crop species to elucidate several aspects of plant physiology and metabolism.

Quercetin inhibits UV irradiation-induced inflammatory cytokine production in primary human keratinocytes by suppressing NF-kappa B pathway

Background: Topical flavonoids, such as quercetin, have been shown to reduce ultraviolet (UV) irradiation-mediated skin damage. However, the mechanisms and signaling pathways involved in this protective effect are not clear. UV irradiation leads to activation of two major signaling pathways, namely nuclear factor kappa B (NF-kappa B) and activator protein-1 (AP-1) pathways. Activation of NF-kappa B pathway by UV irradiation stimulates inflammatory cytokine expression, whereas activation of AP-1 pathway by UV irradiation promotes matrix metalloproteinase (MMP) production. Both pathways contribute to UV irradiation-induced skin damage, such as photoaging and skin tumor formation. Objective: To elucidate the underlying mechanism, we examined the effect of quercetin on UV irradiation induced activation of NF-kappa B and AP-1 pathways. Methods: Primary human keratinocytes, the major skin cell type subjected to physiological solar UV irradiation, were used to study the effects of quercetin on UV irradiation-induced signal transduction pathways. Results: Quercetin decreased UV irradiation-induced NF-kappa B DNA-binding by 80%. Consequently, quercetin suppressed UV irradiation-induced expression of inflammatory cytokines IL-1 beta (similar to 60%), IL-6 (similar to 80%), IL-8 (similar to 76%) and TNF-alpha (similar to 69%). In contrast, quercetin had no effect on UV irradiation activation of three MAP kinases, ERK, JNK, or p38. Accordingly, induction of AP-1 target genes such as MMP-1 and MMP-3 by UV irradiation was not suppressed by quercetin. Conclusion: Our data indicate that the ability of quercetin to block UV irradiation-induced skin inflammation is mediated, at least in part, by its inhibitory effect on NF-kappa B activation and inflammatory cytokine production. (C) 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Molecular cloning reveals that the p160 myb-binding protein is a novel, predominantly nucleolar protein which may play a role in transactivation by Myb

We have previously detected two related murine nuclear proteins, p160 and p67, that can bind to the leucine zipper motif within the negative regulatory domain of the Myb transcription factor. We now describe the molecular cloning of cDNA corresponding to murine p160. The P160 gene is located on mouse chromosome 11, and related sequences are found on chromosomes 1 and 12. The predicted p160 protein is novel, and in agreement with previous studies, we find that the corresponding 4.5-kb mRNA is ubiquitously expressed. We showed that p67 is an N-terminal fragment of p160 which is generated by proteolytic cleavage in certain cell types. The protein encoded by the cloned p160 cDNA and an engineered protein (p67*) comprising the amino-terminal region of p160 exhibit binding specificities for the Myb and Jun leucine zipper regions identical to those of endogenous p160 and p67, respectively. This implies that the Myb-binding site of p160 lies within the N-terminal 580 residues and that the Jun-binding site is C-terminal to this position. Moreover, we show that p67* but not p160 can inhibit transactivation by Myb. Unexpectedly, immunofluorescence studies show that p160 is localized predominantly in the nucleolus. The implications of these results for possible functions of p160 are discussed.

Biomolecular interaction analysis of IFN gamma-induced signaling events in whole-cell lysates: Prevalence of latent STAT1 in high-molecular weight complexes

The basic framework for the JAK/STAT pathway is well documented. Recruitment of latent cytoplasmic STAT transcription factors to tyrosine phosphorylated docking sites on cytokine receptors and their JAK-mediated phosphorylation instigates their translocation to the nucleus and their ability to bind DNA, The biochemical processes underlying recruitment and activation of this pathway have commonly been studied in reconstituted in vitro systems using previously defined recombinant signaling components. We have dissected the Interferon gamma (IFN gamma) signal transduction pathway in crude extracts from wild-type and STAT1-negative mutant cell Lines by real-time BIAcore analysis, size-exclusion (SE) chromatography and immune-detection. The data indicate that in detergent-free cell extracts: (1) the phospho-tyrosine (Y440P)-containing peptide motif of the IFN gamma-receptor ct-chain interacts directly with STAT1, or STAT1 complexes, and no other protein; (2) nonactivated STAT 1 is present in a higher molecular weight complex(es) and, at least for IFN gamma-primed cells, is available for recruitment to the activated IFN gamma-receptor from only a subset of such complexes; (3) activated STAT1 is released from the receptor as a monomer.

Key residues characteristic of GATA N-fingers are recognized by FOG

Protein-protein interactions play significant roles in the control of gene expression. These interactions often occur between small, discrete domains within different transcription factors. In particular, zinc fingers, usually regarded as DNA-binding domains, are now also known to be involved in mediating contacts between proteins. We have investigated the interaction between the erythroid transcription factor GATA-1 and its partner, the 9 zinc finger protein, FOG (Friend of GATA). We demonstrate that this interaction represents a genuine finger-finger contact, which is dependent on zinc coordinating residues within each protein. We map the contact domains to the core of the N-terminal zinc finger of GATA-1 and the 6th zinc finger of FOG. Using a scanning substitution strategy we identify key residues within the GATA-1 N-finger which are required for FOG binding. These residues are conserved in the N-fingers of all GATA proteins known to bind FOG, but are not found in the respective C-fingers, This observation may, therefore, account for the particular specificity of FOG for N-fingers, Interestingly, the key N-finger residues are seen to form a contiguous surface, when mapped onto the structure of the N-finger of GATA-1.

Downregulation of ultraspiracle gene expression delays pupal development in honeybees

Ecdysteroids regulate many aspects of insect physiology after binding to a heterodimer composed of the nuclear hormone receptor proteins ecdysone receptor (EcR) and ultraspiracle (Use). Several lines of evidence have suggested that the latter also plays important roles in mediating the action of juvenile hormone (JH) and, thus, integrates signaling by the two morphogenetic hormones. By using an RNAi approach, we show here that Us p participates in the mechanism that regulates the progression of pupal development in Apis mellifera, as indicated by the observed pupal developmental delay in usp knocked-down bees. Knock-down experiments also suggest that the expression of regulatory genes such as ftz transcription factor 1 (ftz-f1) and juvenile hormone esterase (jhe) depend on Usp. Vitellogenin (vg), the gene coding the main yolk protein in honeybees, does not seem to be under Usp regulation, thus suggesting that the previously observed induction of vg expression by JH during the last stages of pupal development is mediated by yet unknown transcription factor complexes. (C) 2008 Elsevier Ltd. All rights reserved.

Phosphorus overload and PTH induce aortic expression of Runx2 in experimental uraemia

Background. Vascular calcification (VC) is commonly seen in patients with chronic kidney disease (CKD). Elevated levels of phosphate and parathormone (PTH) are considered nontraditional risk factors for VC. It has been shown that, in vitro, phosphate transforms vascular smooth muscle cells (VSMCs) into calcifying cells, evidenced by upregulated expression of runt-related transcription factor 2 (Runx2), whereas PTH is protective against VC. In addition, Runx2 has been detected in calcified arteries of CKD patients. However, the in vivo effect of phosphate and PTH on Runx2 expression remains unknown. Methods. Wistar rats were submitted to parathyroidectomy, 5/6 nephrectomy (Nx) and continuous infusion of 1-34 rat PTH (at physiological or supraphysiological rates) or were sham-operated. Diets varied only in phosphate content, which was low (0.2%) or high (1.2%). Biochemical, histological, immunohistochemistry and immunofluorescence analyses were performed. Results. Nephrectomized animals receiving high-PTH infusion presented VC, regardless of the phosphate intake level. However, phosphate overload and normal PTH infusion induced phenotypic changes in VSMCs, as evidenced by upregulated aortic expression of Runx2. High-PTH infusion promoted histological changes in the expression of osteoprotegerin and type I collagen in calcified arteries. Conclusions. Phosphate, by itself is a potential pathogenic factor for VC. It is of note that phosphate overload, even without VC, was associated with overexpression of Runx2 in VSMCs. The mineral imbalance often seen in patients with CKD should be corrected.

DNA binding-independent transcriptional activation of the vascular endothelial growth factor gene (VEGF) by the Myb oncoprotein

Myb is a key transcription factor that can regulate proliferation, differentiation, and apoptosis, predominantly in the haemopoietic system. Abnormal expression of Myb is associated with a number of cancers, both haemopoietic and non-haemopoietic. In order to better understand the role of Myb in normal and tumorigenic processes, we undertook a cDNA array screen to identify genes that are regulated by this factor. In this way, we identified the gene encoding vascular endothelial growth factor (VEGF) as being potentially regulated by the Myb oncoprotein in myeloid cells. To determine whether this was a direct effect on VEGF gene transcription, we examined the activity of the murine VEGF promoter in the presence of either wild-type (WT) or mutant forms of Myb. It was found that WT Myb was able to activate the VEGF promoter and that a minimal promoter region of 120 bp was sufficient to confer Myb responsiveness. Surprisingly, activation of the VEGF promoter was independent of DNA binding by Myb. This was shown by the use of DNA binding-defective Myb mutants and by mutagenesis of a potential Myb-binding site in the minimal promoter. Mutation of Sp1 sites within this region abolished Myb-mediated regulation of a reporter construct, suggesting that Myb DNA binding-independent activation of VEGF expression occurs via these Sp1 binding elements. Regulation of VEGF production by Myb has implications for the potential role of Myb in myeloid leukaemias and in solid tumours where VEGF may be functioning as an autocrine growth factor. (c) 2006 Elsevier Inc. All rights reserved.

Novel Heterozygous Nonsense GLI2 Mutations in Patients with Hypopituitarism and Ectopic Posterior Pituitary Lobe without Holoprosencephaly

Context: GLI2 is a transcription factor downstream in Sonic Hedgehog signaling, acting early in ventral forebrain and pituitary development. GLI2 mutations were reported in patients with holoprosencephaly (HPE) and pituitary abnormalities. Objective: The aim was to report three novel frameshift/nonsense GLI2 mutations and the phenotypic variability in the three families. Setting: The study was conducted at a university hospital. Patients and Methods: The GLI2 coding region of patients with isolated GH deficiency (IGHD) or combined pituitary hormone deficiency was amplified by PCR using intronic primers and sequenced. Results: Three novel heterozygous GLI2 mutations were identified: c. 2362_2368del p. L788fsX794 (family 1), c. 2081_2084del p. L694fsX722 (family 2), and c. 1138 G > T p. E380X (family 3). All predict a truncated protein with loss of the C-terminal activator domain. The index case of family 1 had polydactyly, hypoglycemia, and seizures, and GH, TSH, prolactin, ACTH, LH, and FSH deficiencies. Her mother and seven relatives harboring the same mutation had polydactyly, including two uncles with IGHD and one cousin with GH, TSH, LH, and FSH deficiencies. In family 2, a boy had cryptorchidism, cleft lip and palate, and GH deficiency. In family 3, a girl had hypoglycemia, seizures, excessive thirst and polyuria, and GH, ACTH, TSH, and antidiuretic hormone deficiencies. Magnetic resonance imaging of four patients with GLI2 mutations and hypopituitarism showed a hypoplastic anterior pituitary and an ectopic posterior pituitary lobe without HPE. Conclusion: We describe three novel heterozygous frameshift or nonsense GLI2 mutations, predicting truncated proteins lacking the activator domain, associated with IGHD or combined pituitary hormone deficiency and ectopic posterior pituitary lobe without HPE. These phenotypes support partial penetrance, variable polydactyly, midline facial defects, and pituitary hormone deficiencies, including diabetes insipidus, conferred by heterozygous frameshift or nonsense GLI2 mutations. (J Clin Endocrinol Metab 95: E384-E391, 2010)