Suppression of black-hole growth by strong outflows at redshifts 5.8-6.6.

Bright quasars, powered by accretion onto billion-solar-mass black holes, already existed at the epoch of reionization, when the Universe was 0.5-1 billion years old1. How these black holes formed in such a short time is the subject of debate, particularly as they lie above the correlation between black-hole mass and galaxy dynamical mass2,3 in the local Universe. What slowed down black-hole growth, leading towards the symbiotic growth observed in the local Universe, and when this process started, has hitherto not been known, although black-hole feedback is a likely driver4. Here we report optical and near-infrared observations of a sample of quasars at redshifts 5.8 ≲ z ≲ 6.6. About half of the quasar spectra reveal broad, blueshifted absorption line troughs, tracing black-hole-driven winds with extreme outflow velocities, up to 17% of the speed of light. The fraction of quasars with such outflow winds at z ≳ 5.8 is ≈2.4 times higher than at z ≈ 2-4. We infer that outflows at z ≳ 5.8 inject large amounts of energy into the interstellar medium and suppress nuclear gas accretion, slowing down black-hole growth. The outflow phase may then mark the beginning of substantial black-hole feedback. The red optical colours of outflow quasars at z ≳ 5.8 indeed suggest that these systems are dusty and may be caught during an initial quenching phase of obscured accretion5.

Aerobic cloacal and pharyngeal bacterial flora in six species of free-living birds.

AIMS: The purpose of this study was to investigate the culturable aerobic pharyngeal and cloacal bacterial flora of free-living birds, to determine the physiological bacterial microbiota, to identify possible interactions between feeding behaviour and the composition of the pharyngeal and cloacal microflora and to investigate the occurrence of pathogenic bacteria. METHODS AND RESULTS: Cloacal and pharyngeal swabs of 167 free-living birds, including water rails (Rallus aquaticus), spotted crakes (Porzana porzana), mute swans (Cygnus olor), barn swallows (Hirundo rustica), reed warblers (Acrocephalus scirpaceus) and black cormorants (Phalacrocorax carbo) from Germany, were cultured to determine the prevalence of aerobic bacteria. Statistical analysis of bacterial findings and feeding behaviour was performed. A widespread soil and water bacteria were isolated, which are expected to be present in the habitat and food. However, some potentially avian- and human-pathogenic bacteria, such as Aeromonas hydrophila, Elizabethkingia meningoseptica, Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli, were also recovered. CONCLUSIONS: Free-living birds of the examined species harbour several environmental bacteria, which could be facultative pathogenic. SIGNIFICANCE AND IMPACT OF THE STUDY: Prevalence of bacteria in healthy free-living birds of the species included in this survey is influenced by environmental and alimentary factors.

The response of gamma interferon activation factor is under developmental control in cells of the macrophage lineage.

Gamma interferon activation factor (GAF) rapidly induces transcriptional activation of gamma interferon (IFN-gamma)-responsive genes. Conversion of the GAF from a latent cytoplasmic to an activated, DNA-binding form is an immediate step in the cellular response to IFN-gamma. The amount of IFN-gamma-activated GAF, measured by exonuclease III protection or gel shift assays, increased strongly upon monocytic differentiation of U937 cells. Activated GAF contained the IFN-responsive 91-kDa protein as its DNA-binding activity in gel shift or exonuclease III assays could be inhibited through direct addition of specific antiserum, and it was not present in p91-immunodepleted extracts. There was a differentiation-induced increase in the amount of nonphosphorylated (latent) p91. Transcription rate measurement demonstrated a strong induction of the p91 gene during monocytic differentiation of U937 cells. The amount of p91 which was rapidly phosphorylated in response to IFN-gamma was found to be much higher in the differentiated cells and suggested a differentiation-controlled increase in the signaling leading to p91 phosphorylation. Concomitantly with a better GAF response, transcriptional activation of IFN-gamma-induced genes and the expression of GAF-dependent, transfected reporter plasmids increased in differentiated U937 monocytes. The promonocyte-monocyte transition also affected the IFN-alpha-responsive transcription factor ISGF-3. Differentiated U937 cells contained more of both the alpha-component p91 and the gamma-component p48, which constitutes the DNA-binding subunit of the complex. Our study thus provides evidence that the synthesis of specific transcription factors can be a regulated event to control the cytokine responsiveness of cells during development.

A factor induced by differentiation signals in cells of the macrophage lineage binds to the gamma interferon activation site.

Rapid transcriptional induction of genes in response to gamma interferon (IFN-gamma) is mediated by the IFN-gamma activation site (GAS) and its cognate protein, the IFN-gamma activation factor (GAF). We describe a GAS-associated, differentiation-induced factor (DIF) as a potential molecular link between the activities of IFN-gamma and of growth and differentiation factors. DIF DNA binding was activated by colony-stimulating factor 1 in murine macrophages and also during tetradecanoyl phorbol acetate-induced differentiation or IFN-gamma treatment in myeloid U937 cells. IFN-gamma activation of DIF decreased significantly upon monocytic differentiation. DIF binding to DNA was inhibited by antiphosphotyrosine antibodies and could be induced by treatment of U937 cells with vanadate. Unlike GAF, DIF-DNA complexes did not contain the 91-kDa protein (p91) from ISGF-3. DIF bound with high affinity to GAS from the promoters of the IFP 53/tryptophanyl-tRNA synthetase and Fc gamma RI genes, intermediate affinity to the Ly6A/E GAS, and low affinity to the guanylate-binding protein GAS. DIF may belong to a family of cytokine- or growth factor-induced factors binding with variable affinities to GAS-related elements: the interleukin-6-responsive acute-phase response factor associated with GAS from different IFN-inducible promoters but with a different preference of binding compared with DIF. The sis-inducible element of the c-fos promoter bound GAF but not DIF. However, the sis-inducible element could be changed by point mutation to compete for GAF and DIF binding. Our data show DIF to be a novel DNA-binding protein which is activated in response to differentiating signals. Moreover, they suggest that a family of cytokine- or growth factor-regulated proteins integrates and coordinates the responses to cytokines and to growth and differentiation factors by binding to GAS-related elements.

MondoA, a novel basic helix-loop-helix-leucine zipper transcriptional activator that constitutes a positive branch of a max-like network.

Max is a common dimerization partner for a family of transcription factors (Myc, Mad [or Mxi]), and Mnt [or Rox] proteins) that regulate cell growth, proliferation, and apoptosis. We recently characterized a novel Max-like protein, Mlx, which interacts with Mad1 and Mad4. Here we describe the cloning and functional characterization of a new family of basic helix-loop-helix-leucine zipper heterodimeric partners for Mlx termed the Mondo family. MondoA forms homodimers weakly and does not interact with Max or members of the Myc or Mad families. MondoA and Mlx associate in vivo, and surprisingly, they are localized primarily to the cytoplasm of cultured mammalian cells. Treatment of cells with the nuclear export inhibitor leptomycin B results in the nuclear accumulation of MondoA and Mlx, demonstrating that they shuttle between the cytoplasmic and nuclear compartments rather than having exclusively cytoplasmic localization. MondoA preferentially forms heterodimers with Mlx, and this heterocomplex can bind to, and activate transcription from, CACGTG E-boxes when targeted to the nucleus via a heterologous nuclear localization signal. The amino termini of the Mondo proteins are highly conserved among family members and contain separable and autonomous cytoplasmic localization and transcription activation domains. Therefore, Mlx can mediate transcriptional repression in conjunction with the Mad family and can mediate transcriptional activation via the Mondo family. We propose that Mlx, like Max, functions as the center of a transcription factor network.

Differentiation-regulated serine phosphorylation of STAT1 promotes GAF activation in macrophages.

Gamma interferon (IFN-gamma), a macrophage-activating cytokine, modulates gene expression through the activity of a transcription factor designated IFN-gamma activation factor (GAF). GAF is formed after phosphorylation on tyrosine and dimerization of the 91-kDa protein STAT1. We have recently reported that differentiation of the promonocytic cell line U937 into monocytes increases the amount of cellular GAF after IFN-gamma treatment and at the same time increases the phosphorylation of STAT1. Here we show that activation of the JAK family kinases, which are instrumental in mediating STAT1 phosphorylation on tyrosine, did not increase upon monocytic U937 differentiation. Consistent with this finding, levels of STAT1 tyrosine phosphorylation were virtually identical in promonocytic and monocytic U937 cells. Analysis of STAT1 phosphoamino acids and mapping of phosphopeptides showed an IFN-gamma-dependent increase in Ser phosphorylation in differentiated cells. Analyses of STAT1 isoforms by two-dimensional gel electrophoresis demonstrated a differentiation-induced shift toward more acidic isoforms. All isoforms were equally sensitive to subsequent tyrosine phosphorylation, as indicated by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility shift typical for tyrosine-phosphorylated STAT1. Consistent with the importance of Ser phosphorylation for high-affinity binding to the IFN-gamma activation site sequence, phosphatase 2A treatment strongly reduced the formation of IFN-gamma activation site-GAF complexes in an electrophoretic mobility shift assay. Our data indicate that the activity of GAF is modulated by STAT1 serine kinases/phosphatases and suggest that this mechanism is employed in the developmental control of macrophage responsiveness to IFN-gamma.

A role for STAT family transcription factors in myeloid differentiation.

STAT family transcription factors regulate gene expression in response to a wide variety of cytokines. A transcription factor designated differentiation-induced factor (DIF), activated by treatment of myeloid cells with the differentiating agents interferon-gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), colony-stimulating factor-1 (CSF-1) or during phorbol ester-induced differentiation, was characterized as a 112kDa protein related to, but not identical with known isoforms of STAT 5. Taken together with previously published results, our data suggest an important function for members of the STAT 5 subfamily in regulating gene expression during the process of myeloid differentiation.

Colony-stimulating factors and interferon-gamma activate a protein related to MGF-Stat 5 to cause formation of the differentiation-induced factor in myeloid cells.

The Jak-Stat pathway of intracellular signals is used by growth factor- and cytokine receptors to induce gene transcription. We have recently reported that differentiation of myeloid cells, induced by phorbol ester, interferon-gamma (IFN-gamma) or colony-stimulating factor-1 (CSF-1) is accompanied by the activation of the differentiation-induced factor (DIF). Activated DIF specifically associates with a subclass of gamma-interferon activation site (GAS)-like DNA elements. We now report that GM-CSF, which like CSF-1 promotes the generation of mature macrophages, activates DIF. No activation was observed after treatment with the granulocyte growth and differentiation factor G-CSF. Antibodies raised against a Stat family protein, designated mammary gland factor-Stat 5 (MGF-Stat 5), reacted with DIF induced by either CSF-1, GM-CSF or IFN-gamma. Antisera to other known Stats were without effect on the DIF complex in electrophoretic mobility shift assays (EMSA). A 112 kDa protein could be isolated from either GM-CSF- or IFN-gamma-treated cells by GAS oligonucleotide precipitation. This protein reacted with antibodies to both MGF-Stat 5 and phosphotyrosine. MGF-Stat 5 and closely related proteins thus define a subfamily of Stat transcription factors that are present in a variety of cell types and are required for the onset of immediate gene expression in response to differentiating stimuli.

c-Jun and the transcriptional control of neuronal apoptosis.

There has been considerable interest in the molecular mechanisms of apoptosis in mammalian neurons because this form of neuronal cell death is important for the normal development of the nervous system and because inappropriate neuronal apoptosis may contribute to the pathology of human neurodegenerative diseases. The aim of recent research has been to identify the key components of the cell death machinery in neurons and understand how the cell death programme is regulated by intracellular signalling pathways activated by the binding of neurotrophins or death factors to specific cell surface receptors. The aim of this commentary was to review research that has investigated the role of the Jun N-terminal kinase (JNK)/c-Jun signalling pathway in neuronal apoptosis, focusing in particular on work carried out with developing sympathetic neurons. Experiments with sympathetic neurons cultured in vitro, as well as with cerebellar granule neurons and differentiated PC12 cells, have demonstrated that JNK/c-Jun signalling can promote apoptosis following survival factor withdrawal. In addition, experiments with Jnk(-/-) knockout mice have provided evidence that Jnk3 may be required for apoptosis in the hippocampus in vivo following injection of kainic acid, an excitotoxin, and that Jnk1 and Jnk2 are required for apoptosis in the developing embryonic neural tube. However, in the embryonic forebrain, Jnk1 and Jnk2 have the opposite function and are necessary for the survival of developing cortical neurons. These results suggest that JNKs and c-Jun are important regulators of the cell death programme in the mammalian nervous system, but that their biological effects depend on the neuronal type and stage of development.

Activity of Stat family transcription factors is developmentally controlled in cells of the macrophage lineage.

Stat family transcription factors are activated in response to a variety of cytokines to bind to a class of DNA elements termed gamma interferon activation site (GAS)-like elements. Here we investigate two GAS-binding transcription factors, the gamma-interferon activation factor (GAF) and the differentiation-induced factor (DIF) that are activated by interferon-gamma (IFN-gamma) in U937 cells. Treatment of U937 cells with phorbol ester (TPA) induces differentiation from a promonocyte into a monocyte stage of macrophage development. Monocytic differentiation led to an increased transcriptional response of GAS-containing genes to IFN-gamma. TPA treatment also caused a profound change in the IFN-gamma activation of GAF and DIF. GAF DNA-binding activity was activated much better in the monocyte stage and the GAF constituent Stat 1 showed increased phosphorylation. In contrast, DIF activation by IFN-gamma was found in promonocytes but was virtually absent in monocytes. Moreover, DIF activation was observed during TPA-induced monocytic differentiation and after treatment of macrophages with the macrophage differentiation factor CSF-1. Our data suggest DIF to be part of a developmental program leading to terminal macrophage differentiation and GAF to be a transcription factor bringing about the stronger activation response of mature macrophages to IFN-gamma.

Effects of barium on stimulus-induced rises in [K+]o in juvenile rat hippocampal area CA1.

Immature glia may not be able to buffer K+ ions released during neuronal activity. Therefore, we investigated entorhinal-hippocampal slices of juvenile rats (ages P15-18 and P22-26) using a perfusion medium containing 2 mM BaCl2 in order to block glial inward rectifying and leak potassium channels. In contrast to adult animals, rises in [K+]o in slices from juvenile animals elicited by repetitive alvear stimulation were not augmented by Ba2+. Ba2+ effects on fast field potentials, slow field potentials and the applied current sink source distribution were roughly similar as in adult rats. We conclude that the capacity to buffer large quantities of K+ ions by mechanisms involving Ba2+-sensitive K+ channels has not yet developed in juveniles.

Effects of barium on stimulus induced changes in extracellular potassium concentration in area CA1 of hippocampal slices from normal and pilocarpine-treated epileptic rats.

Laminar profiles of rises in [K+]o and slow field potentials induced by alvear stimulation were recorded in area CA1 of hippocampal slices from control and pilocarpine-treated rats in absence and presence of Ba2+. In control animals, Ba2+ augmented rises in [K+]o in stratum pyramidale (SP) as well as in stratum radiatum (SR). In pilocarpine-treated animals an augmentation of rises in [K+]o was restricted to SP and its immediate vicinity. Moreover, the effect of Ba2+ in SP was small or missing in eight out of 15 slices of pilocarpine-treated animals. In these slices laminar profiles of rises in [K+]o were not affected by Ba2+. It is suggested that spatial K+-buffering is reduced in area CA1 of epileptic animals.

Efficacy of vital home bleaching.

The nightguard vital bleaching technique has become a very common and accepted procedure for many dental practitioners. Much has been written about the procedure's history, safety and technique; little has been done to quantify the specific color changes of teeth resulting it. This clinical study attempted to give a numerical value to the color change through the use of a light transmission densitometer. The maxillary teeth of twenty-five patients were treated as the control. A significant whitening was observed for a majority of the patients during the four-week duration of the study.

Alcohol-induced Purkinje cell loss with a single binge exposure in neonatal rats: a stereological study of temporal windows of vulnerability.

Previous research has shown that the early neonatal period of rats is one of enhanced vulnerability to cerebellar Purkinje cell loss associated with binge-like alcohol exposure, with a prominent sensitive period during the first neonatal week. In this study, an unbiased count of the total number of Purkinje cells was obtained using the stereological optical fractionator, in groups of rats given a single binge-like alcohol exposure either during the most vulnerable neonatal period [postnatal day (PD) 4] or during a later, less vulnerable period (PD 9). Using artificial rearing methods, rats were given 6.6 g/kg of alcohol either on PD 4 or on PD 9, delivered as a 15% (v/v) solution in milk formula on two consecutive feedings of the designated day. Control groups included an artificially reared gastrostomy control and a normally reared suckle control. The mean peak blood alcohol concentrations were not different between the PD 4 and PD 9 alcohol groups, averaging 374 and 347 mg/dl, respectively. The rats were perfused on PD 27. A uniform random sample of sections was obtained from serial frozen sections through the cerebellum, stained with thionin, and Purkinje cells were counted from a uniform random sample of locations on each section with the three-dimensional optical fractionator. The number of Purkinje cells in the suckle control and gastrostomy control groups did not differ from each other, averaging 3.94 (+/- 0.19) and 3.58 (+/- 0.22) x 10(5) cells, respectively. Binge exposure on PD 4 induced significant cell loss (mean of 2.05 +/- 0.20 x (10(5) Purkinje cells), whereas binge exposure on PD 9 did not induce significant Purkinje cell loss (3.70 +/- 0.39 x 10(5) Purkinje cells). These findings confirm that a single neonatal binge alcohol exposure produces pathological Purkinje cell loss, provided that it occurs during the period of enhanced vulnerability coinciding with the early stages of dendritic outgrowth.

Role of a distal promoter element in the S-phase control of the human H1.2 histone gene transcription.

The expression of one of the human main type H1 histone genes (termed H1.2) appears to be regulated by several trans-acting factors. Upstream of consensus regulatory regions, such as the TATA-, CCAAT- and H1-box (AAACACA) sequences, a crucial control site is located between nucleotide positions -536 and -412 (relative to the ATG initiation site). Removal of this promoter portion causes in chloramphenicol acetyl transferase reporter gene constructs a loss of the S-phase control function of the H1.2 promoter in HeLa cells. Electrophoretic mobility-shift assay and DNase I footprinting analysis suggest that the H1-box variant AAACAGA is a potential control element within the distal promoter region.

Constitutive STAT1 tyrosine phosphorylation in U937 monocytes overexpressing the TYK2 protein tyrosine kinase does not induce gene transcription.

Janus kinase (JAK) family protein tyrosine kinases are constituents of a signaling path leading to tyrosine phosphorylation and activation of signal transducer and activator of transcription (STAT) family transcription factors. IFN-alpha activates two JAK family protein tyrosine kinases (TYK2 and JAK1) and two STAT family proteins (STAT1 and STAT2). We have generated a line of U937 promonocytes expressing a tyk2 transgene. 12-O-Tetradecanoylphorbol-13-acetate-mediated differentiation into monocytes resulted in transgene induction and both overexpression and constitutive activation of the kinase. TYK2 protein in the transgenic line was found predominantly in a membrane fraction. Coprecipitation experiments demonstrated an association of constitutively tyrosine-phosphorylated TYK2 with the IFN-alpha receptor 1 chain. TYK2 activity led to an IFN-alpha-independent appearance of tyrosine-phosphorylated STAT1 but not STAT2 or JAK1 proteins. Consistent with this, TYK2 activity also caused constitutive activation of the IFN-alpha-responsive transcription factor IFN-alpha activation factor, a dimer of tyrosine-phosphorylated STAT1, but not of the IFN-alpha-responsive transcription factor IFN-stimulated gene factor 3, a heterotrimer of tyrosine-phosphorylated STAT1 and STAT2 in association with a M(r) 48,000 DNA-binding subunit. Expression of STAT1 target genes was not observed in TYK2-overexpressing cells. Our results suggest that in addition to activated TYK2, there is a requirement for additional, IFN-alpha-dependent signals for the phosphorylation of STAT2 and the generation of IFN-stimulated gene factor 3 as well as for the conversion of tyrosine-phosphorylated STAT1 into transcriptionally active IFN-alpha activation factor.

Role of the Jun kinase pathway in the regulation of c-Jun expression and apoptosis in sympathetic neurons.

When deprived of nerve growth factor (NGF), developing sympathetic neurons die by apoptosis. This death is associated with an increase in the level of c-Jun protein and is blocked by expression of a c-Jun dominant negative mutant. Here we have investigated whether NGF withdrawal activates Jun kinases, a family of stress-activated protein kinases that can stimulate the transcriptional activity of c-Jun by phosphorylating serines 63 and 73 in the transactivation domain and which can activate c-jun gene expression. We found that sympathetic neurons contained high basal levels of Jun kinase activity that increased further after NGF deprivation. In contrast, p38 kinase, another stress-activated protein kinase that can also stimulate c-jun gene expression, was not activated after NGF withdrawal. Consistent with Jun kinase activation, we found using a phospho-c-Jun-specific antibody that c-Jun was phosphorylated on serine 63 after NGF withdrawal. Furthermore, expression of a constitutively active form of MEK kinase 1 (MEKK1), which strongly activates the Jun kinase pathway, increased c-Jun protein levels and c-Jun phosphorylation and induced apoptosis in the presence of NGF. This death could be prevented by co-expression of SEKAL, a dominant negative mutant of SAPK/ERK kinase 1 (SEK1), an activator of Jun kinase that is a target of MEKK1. In contrast, expression of SEKAL alone did not prevent c-Jun expression, increases in c-Jun phosphorylation, or cell death after NGF withdrawal. Thus, activation of Jun kinase and increases in c-Jun phosphorylation and c-Jun protein levels occur at the same time after NGF withdrawal, but c-Jun levels and phosphorylation are regulated by an SEK1-independent pathway.

Mlx, a novel Max-like BHLHZip protein that interacts with the Max network of transcription factors.

Mad:Max heterodimers oppose the growth-promoting action of Myc:Max heterodimers by recruiting the mSin3-histone deacetylase (mSin3. HDAC) complex to DNA and functioning as potent transcriptional repressors. There are four known members of the Mad family that are indistinguishable in their abilities to interact with Max, bind DNA, repress transcription, and block Myc + Ras co-transformation. To investigate functional differences between Mad family proteins, we have identified additional proteins that interact with this family. Here we present the identification and characterization of the novel basic-helix-loop-helix zipper protein Mlx (Max-like protein x), which is structurally and functionally related to Max. The similarities between Mlx and Max include 1) broad expression in many tissues, 2) long protein half-life, and 3) formation of heterodimers with Mad family proteins that are capable of specific CACGTG binding. We show that transcriptional repression by Mad1:Mlx heterodimers is dependent on dimerization, DNA binding, and recruitment of the mSin3A.HDAC corepressor complex. In contrast with Max, Mlx interacts only with Mad1 and Mad4. Together, these findings suggest that Mlx may act to diversify Mad family function by its restricted association with a subset of the Mad family of transcriptional repressors.

A 13-amino acid amphipathic alpha-helix is required for the functional interaction between the transcriptional repressor Mad1 and mSin3A.

Members of the Mad family of bHLHZip proteins heterodimerize with Max and function to repress the transcriptional and transforming activities of the Myc proto-oncogene. Mad:Max heterodimers repress transcription by recruiting a large multi-protein complex containing the histone deacetylases, HDAC1 and HDAC2, to DNA. The interaction between Mad proteins and HDAC1/2 is mediated by the corepressor mSin3A and requires sequences at the amino terminus of the Mad proteins, termed the SID, for Sin3 interaction domain, and the second of four paired amphipathic alpha-helices (PAH2) in mSin3A. To better understand the requirements for the interaction between the SID and PAH2, we have performed mutagenesis and structural studies on the SID. These studies show that amino acids 8-20 of Mad1 are sufficient for SID:PAH2 interaction. Further, this minimal 13-residue SID peptide forms an amphipathic alpha-helix in solution, and residues on the hydrophobic face of the SID helix are required for interaction with PAH2. Finally, the minimal SID can function as an autonomous and portable repression domain, demonstrating that it is sufficient to target a functional mSin3A/HDAC corepressor complex.