Mobilization of the A-kinase N-myristate through an isoform-specific intermolecular switch

Although the catalytic (C) subunit of cAMP-dependent protein kinase is N-myristylated, it is a soluble protein, and no physiological role has been identified for its myristyl moiety. To determine whether the interaction of the two regulatory (R) subunit isoforms (RI and RII) with the N-myristylated C subunit affects its ability to target membranes, the effect of N-myristylation and the RI and RII subunit isoforms on C subunit binding to phosphatidylcholine/phosphatidylserine liposomes was examined. Only the combination of N-myristylation and RII subunit interaction produced a dramatic increase in the rate of liposomal binding. To assess whether the RII subunit also increased the conformational flexibility of the C subunit N terminus, the effect of N-myristylation and the RI and RII subunits on the rotational freedom of the C subunit N terminus was measured. Specifically, fluorescein maleimide was conjugated to Cys-16 in the N-terminal domain of a K16C mutant of the C subunit, and the time-resolved emission anisotropy was determined. The interaction of the RII subunit, but not the RI subunit, significantly increased the backbone flexibility around the site of mutation and labeling, strongly suggesting that RII subunit binding to the myristylated C subunit induced a unique conformation of the C subunit that is associated with an increase in both the N-terminal flexibility and the exposure of the N-myristate. RII subunit thus appears to serve as an intermolecular switch that disrupts of the link between the N-terminal and core catalytic domains of the C subunit to expose the N-myristate and poise the holoenzyme for interaction with membranes.

Ionizing radiation and short wavelength UV activate NF-κB through two distinct mechanisms

We examined the mechanisms by which two different types of photonic radiation, short wavelength UV (UV-C) and γ radiation, activate transcription factor NF-κB. Exposure of mammalian cells to either form of radiation resulted in induction with similar kinetics of NF-κB DNA binding activity, nuclear translocation of its p65(RelA) subunit, and degradation of the major NF-κB inhibitor IκBα. In both cases, induction of NF-κB activity was attenuated by proteasome inhibitors and a mutation in ubiquitin-activating enzyme, suggesting that both UV-C and γ radiation induce degradation of IκBs by means of the ubiquitin/proteasome pathway. However, although the induction of IκBα degradation by γ rays was dependent on its phosphorylation at Ser-32 and Ser-36, UV-C-induced IκBα degradation was not dependent on phosphorylation of these residues. Even the “super repressor” IκBα mutant, which contains alanines at positions 32 and 36, was still susceptible to UV-C-induced degradation. Correspondingly, we found that γ radiation led to activation of IKK, the protein kinase that phosphorylates IκBα at Ser-32 and Ser-36, whereas UV-C radiation did not. Furthermore, expression of a catalytically inactive IKKβ mutant prevented NF-κB activation by γ radiation, but not by UV-C. These results indicate that γ radiation and UV-C activate NF-κB through two distinct mechanisms.

Withdrawal of IL-7 induces Bax translocation from cytosol to mitochondria through a rise in intracellular pH

IL-7 functions as a trophic factor during T lymphocyte development by a mechanism that is partly based on the induction of Bcl-2, which protects cells from apoptosis. Here we report a mechanism by which cytokine withdrawal activates the prodeath protein Bax. On loss of IL-7 in a dependent cell line, Bax protein translocated from the cytosol to the mitochondria, where it integrated into the mitochondrial membrane. This translocation was attributable to a conformational change in the Bax protein itself. We show that a rise in intracellular pH preceded mitochondrial translocation and triggered the change in Bax conformation. Intracellular pH in the IL-7-dependent cells rose steadily to peak over pH 7.8 by 6 hr after cytokine withdrawal, paralleling the time point of Bax translocation (a similar alkalinization and Bax translocation was also observed after IL-3 withdrawal from a dependent cell line). The conformation of Bax was directly altered by pH of 7.8 or higher and was demonstrated by increased protease sensitivity, exposure of N terminus epitopes, and exposure of a hydrophobic domain in the C terminus. Eliminating charged amino acids at the C or N termini of Bax induced a conformational change similar to that induced by raising pH, implicating these residues in the pH effect. Therefore, we have shown that by either cytokine withdrawal, experimental manipulation of pH, or site-directed mutagenesis, Bax protein changes conformation, exposing membrane-seeking domains, thereby inducing mitochondrial translocation and initiating the cascade of events leading to apoptotic death.

Convergence of two repressors through heterodimer formation of androgen receptor and testicular orphan receptor-4: A unique signaling pathway in the steroid receptor superfamily

The androgen receptor (AR) binds to androgen response elements and regulates target genes via a mechanism involving coregulators. Here we demonstrate that the AR can interact with the testicular orphan receptor-4 (TR4) and function as a repressor to down-regulate the TR4 target genes by preventing the TR4 binding to its target DNA. Interestingly, the heterodimerization of AR and TR4 also allows TR4 to repress AR target gene expression. Simultaneous exposure to both receptors therefore could result in bidirectional suppression of their target genes. Together, these data demonstrate that the coupling of two different receptors, through the heterodimerization of AR and TR4, is a unique signaling pathway in the steroid receptor superfamily, which may facilitate further understanding of the complicated androgen action in prostate cancer or libido.

Neonatal lead exposure impairs development of rodent barrel field cortex

Childhood exposure to low-level lead can permanently reduce intelligence, but the neurobiologic mechanism for this effect is unknown. We examined the impact of lead exposure on the development of cortical columns, using the rodent barrel field as a model. In all areas of mammalian neocortex, cortical columns constitute a fundamental structural unit subserving information processing. Barrel field cortex contains columnar processing units with distinct clusters of layer IV neurons that receive sensory input from individual whiskers. In this study, rat pups were exposed to 0, 0.2, 1, 1.5, or 2 g/liter lead acetate in their dam's drinking water from birth through postnatal day 10. This treatment, which coincides with the development of segregated columns in the barrel field, produced blood lead concentrations from 1 to 31 μg/dl. On postnatal day 10, the area of the barrel field and of individual barrels was measured. A dose-related reduction in barrel field area was observed (Pearson correlation = −0.740; P < 0.001); mean barrel field area in the highest exposure group was decreased 12% versus controls. Individual barrels in the physiologically more active caudoventral group were affected preferentially. Total cortical area measured in the same sections was not altered significantly by lead exposure. These data support the hypothesis that lead exposure may impair the development of columnar processing units in immature neocortex. We demonstrate that low levels of blood lead, in the range seen in many impoverished inner-city children, cause structural alterations in a neocortical somatosensory map.

Preferential Zn2+ influx through Ca2+-permeable AMPA/kainate channels triggers prolonged mitochondrial superoxide production

Synaptically released Zn2+ can enter and cause injury to postsynaptic neurons. Microfluorimetric studies using the Zn2+-sensitive probe, Newport green, examined levels of [Zn2+]i attained in cultured cortical neurons on exposure to N-methyl-d-asparte, kainate, or high K+ (to activate voltage-sensitive Ca2+ channels) in the presence of 300 μM Zn2+. Indicating particularly high permeability through Ca2+-permeable α-amino3-hydroxy-5-methyl-4-isoxazolepropionic-acid/kainate (Ca-A/K) channels, micromolar [Zn2+]i rises were observed only after kainate exposures and only in neurons expressing these channels [Ca-A/K(+) neurons]. Further studies using the oxidation-sensitive dye, hydroethidine, revealed Zn2+-dependent reactive oxygen species (ROS) generation that paralleled the [Zn2+]i rises, with rapid oxidation observed only in the case of Zn2+ entry through Ca-A/K channels. Indicating a mitochondrial source of this ROS generation, hydroethidine oxidation was inhibited by the mitochondrial electron transport blocker, rotenone. Additional evidence for a direct interaction between Zn2+ and mitochondria was provided by the observation that the Zn2+ entry through Ca-A/K channels triggered rapid mitochondrial depolarization, as assessed by using the potential-sensitive dye tetramethylrhodamine ethylester. Whereas Ca2+ influx through Ca-A/K channels also triggers ROS production, the [Zn2+]i rises and subsequent ROS production are of more prolonged duration.

The neuroprotective effect of pituitary adenylate cyclase-activating polypeptide on cerebellar granule cells is mediated through inhibition of the CED3-related cysteine protease caspase-3/CPP32

Caspase-3 knockout mice exhibit thickening of the internal granule cell layer of the cerebellum. Concurrently, it has been shown that intracerebral injection of pituitary adenylate cyclase-activating polypeptide (PACAP) induces a transient increase of the thickness of the cerebellar cortex. In the present study, we have investigated the possible effect of PACAP on caspase activity in cultured cerebellar granule cells from 8-day-old rat. Incubation of granule neurons with PACAP for 24 h promoted cell survival and prevented DNA fragmentation. Exposure of cerebellar granule cells to the specific caspase-3 inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethylketone (Z-DEVD-FMK) for 24 h markedly enhanced cell survival and inhibited apoptotic cell death. Time-course studies revealed that PACAP causes a prolonged inhibition of caspase-3 activity without affecting caspase-1. Administration of graded concentrations of PACAP for 3 h induced a dose-dependent inhibition of caspase-3 activity. Incubation of granule cells with both dibutyryl-cAMP (dbcAMP) and phorbol 12-myristate 13-acetate (PMA) mimicked the inhibitory effect of PACAP on caspase-3. Cotreatment of cultured neurons with the protein kinase A inhibitor H89 and the protein kinase C inhibitor chelerythrine abrogated the effect of PACAP on caspase-3 activity. In contrast, the ERK kinase inhibitor U0126 did not affect the action of PACAP on caspase-3 activity. These data demonstrate that PACAP prevents cerebellar granule neurons from apoptotic cell death through a protein kinase A- and protein kinase C-dependent inhibition of caspase-3 activity.

A cellular defense pathway regulating transcription through poly(ADP-ribosyl)ation in response to DNA damage

DNA damage is known to trigger key cellular defense pathways such as those involved in DNA repair. Here we provide evidence for a previously unrecognized pathway regulating transcription in response to DNA damage and show that this regulation is mediated by the abundant nuclear enzyme poly(ADP-ribose) polymerase. We found that poly(ADP-ribose) polymerase reduced the rate of transcription elongation by RNA polymerase II, suggesting that poly(ADP-ribose) polymerase negatively regulates transcription, possibly through the formation of poly(ADP-ribose) polymerase–RNA complexes. In damaged cells, poly(ADP-ribose) polymerase binds to DNA breaks and automodifies itself in the presence of NAD+, resulting in poly(ADP-ribose) polymerase inactivation. We found that automodification of poly(ADP-ribose) polymerase in response to DNA damage resulted in the up-regulation of transcription, presumably because automodified poly(ADP-ribose) polymerase molecules were released from transcripts, thereby relieving the block on transcription. Because agents that damage DNA damage RNA as well, up-regulation of RNA synthesis in response to DNA damage may provide cells with a mechanism to compensate for the loss of damaged transcripts and may be critical for cell survival after exposure to DNA-damaging agents.

Molecular evolution of two vertebrate aryl hydrocarbon (dioxin) receptors (AHR1 and AHR2) and the PAS family

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor through which halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cause altered gene expression and toxicity. The AHR belongs to the basic helix–loop–helix/Per-ARNT-Sim (bHLH-PAS) family of transcriptional regulatory proteins, whose members play key roles in development, circadian rhythmicity, and environmental homeostasis; however, the normal cellular function of the AHR is not yet known. As part of a phylogenetic approach to understanding the function and evolutionary origin of the AHR, we sequenced the PAS homology domain of AHRs from several species of early vertebrates and performed phylogenetic analyses of these AHR amino acid sequences in relation to mammalian AHRs and 24 other members of the PAS family. AHR sequences were identified in a teleost (the killifish Fundulus heteroclitus), two elasmobranch species (the skate Raja erinacea and the dogfish Mustelus canis), and a jawless fish (the lamprey Petromyzon marinus). Two putative AHR genes, designated AHR1 and AHR2, were found both in Fundulus and Mustelus. Phylogenetic analyses indicate that the AHR2 genes in these two species are orthologous, suggesting that an AHR gene duplication occurred early in vertebrate evolution and that multiple AHR genes may be present in other vertebrates. Database searches and phylogenetic analyses identified four putative PAS proteins in the nematode Caenorhabditis elegans, including possible AHR and ARNT homologs. Phylogenetic analysis of the PAS gene family reveals distinct clades containing both invertebrate and vertebrate PAS family members; the latter include paralogous sequences that we propose have arisen by gene duplication early in vertebrate evolution. Overall, our analyses indicate that the AHR is a phylogenetically ancient protein present in all living vertebrate groups (with a possible invertebrate homolog), thus providing an evolutionary perspective to the study of dioxin toxicity and AHR function.

Overexpression of Iron Superoxide Dismutase in Transformed Poplar Modifies the Regulation of Photosynthesis at Low CO2 Partial Pressures or Following Exposure to the Prooxidant Herbicide Methyl Viologen1

Chloroplast-targeted overexpression of an Fe superoxide dismutase (SOD) from Arabidopsis thaliana resulted in substantially increased foliar SOD activities. Ascorbate peroxidase, glutathione reductase, and monodehydroascorbate reductase activities were similar in the leaves from all of the lines, but dehydroascorbate reductase activity was increased in the leaves of the FeSOD transformants relative to untransformed controls. Foliar H2O2, ascorbate, and glutathione contents were comparable in all lines of plants. Irradiance-dependent changes in net CO2 assimilation and chlorophyll a fluorescence quenching parameters were similar in all lines both in air (21% O2) and at low (1%) O2. CO2-response curves for photosynthesis showed similar net CO2-exchange characteristics in all lines. In contrast, values of photochemical quenching declined in leaves from untransformed controls at intercellular CO2 (Ci) values below 200 μL L−1 but remained constant with decreasing Ci in leaves of FeSOD transformants. When the O2 concentration was decreased from 21 to 1%, the effect of FeSOD overexpression on photochemical quenching at limiting Ci was abolished. At high light (1000 μmol m−2 s−1) a progressive decrease in the ratio of variable (Fv) to maximal (Fm) fluorescence was observed with decreasing temperature. At 6oC the high-light-induced decrease in the Fv/Fm ratio was partially prevented by low O2 but values were comparable in all lines. Methyl viologen caused decreased Fv/Fm ratios, but this was less marked in the FeSOD transformants than in the untransformed controls. These observations suggest that the rate of superoxide dismutation limits flux through the Mehler-peroxidase cycle in certain conditions.

Autocrine signaling through ATP release represents a novel mechanism for cell volume regulation.

Recovery of cell volume in response to osmotic stress is mediated in part by increases in the Cl- permeability of the plasma membrane. These studies evaluate the hypothesis that ATP release and autocrine stimulation of purinergic (P2) receptors couple increases in cell volume to opening of Cl- channels. In HTC rat hepatoma cells, swelling induced by hypotonic exposure increased membrane Cl- current density to 44.8 +/- 7.1 pA/pF at -80 mV. Both the rate of volume recovery and the increase in Cl- permeability were inhibited in the presence of the ATP hydrolase apyrase (3 units/ml) or by exposure to the P2 receptor blockers suramin and Reactive Blue 2 (10-100 microM). Cell swelling also stimulated release of ATP. Hypotonic exposure increased the concentration of ATP in the effluent of perfused cells by 170 +/- 36 nM in the presence of a nucleotidase inhibitor (P < 0.01). In whole-cell recordings with ATP as the charge carrier, cell swelling increased membrane current density approximately 30-fold to 16.5 +/- 10.4 pA/pF. These findings indicate that increases in cell volume lead to efflux of ATP through opening of a conductive pathway consistent with a channel, and that extracellular ATP is required for recovery from swelling. ATP may function as an autocrine factor that couples increases in cell volume to opening of Cl- channels through stimulation of P2 receptors.

Oxygen causes fetal pulmonary vasodilation through activation of a calcium-dependent potassium channel.

At birth, pulmonary vasodilation occurs as air-breathing life begins. The mechanism of O2-induced pulmonary vasodilation is unknown. We proposed that O2 causes fetal pulmonary vasodilation through activation of a calcium-dependent potassium channel (KCa) via a cyclic nucleotide-dependent kinase. We tested this hypothesis in hemodynamic studies in acutely prepared fetal lambs and in patch-clamp studies on resistance fetal pulmonary artery smooth muscle cells. Fetal O2 tension (PaO2) was increased by ventilating the ewe with 100% O2, causing fetal total pulmonary resistance to decrease from 1.18 +/- 0.14 to 0.41 +/- 0.03 mmHg per ml per min. Tetraethylammonium and iberiotoxin, preferential KCa-channel inhibitors, attenuated O2-induced fetal pulmonary vasodilation, while glibenclamide, an ATP-sensitive K+-channel antagonist, had no effect. Treatment with either a guanylate cyclase antagonist (LY83583) or cyclic nucleotide-dependent kinase inhibitors (H-89 and KT 5823) significantly attenuated O2-induced fetal pulmonary vasodilation. Under hypoxic conditions (PaO2 = 25 mmHg), whole-cell K+-channel currents (Ik) were small and were inhibited by 1 mM tetraethylammonium or 100 nM charybdotoxin (CTX; a specific KCa-channel blocker). Normoxia (PaO2 = 120 mmHg) increased Ik by more than 300%, and this was reversed by 100 nM CTX. Nitric oxide also increased Ik. Resting membrane potential was -37.2 +/- 1.9 mV and cells depolarized on exposure to CTX, while hyperpolarizing in normoxia. We conclude that O2 causes fetal pulmonary vasodilation by stimulating a cyclic nucleotide-dependent kinase, resulting in KCa-channel activation, membrane hyperpolarization, and vasodilation.

Encapsulação de colecalciferol (vitamina D3) por spray chilling

A indústria de alimentos está constantemente desenvolvendo produtos que fornecem, além de nutrientes, benefícios adicionais à saúde, tais como os enriquecidos com vitaminas. A vitamina D3 (colecalciferol) é sintetizada na pele durante a exposição da luz solar, controla a homeostase de cálcio e fósforo, metabolismo ósseo, pressão arterial e reabsorção renal de cálcio. O processo de microencapsulação vem sendo bastante aplicado em alimentos e um dos objetivos principais é o controle da liberação do agente ativo no momento e local desejado. A tecnologia de spray chilling é interessante para a microencapsulação de vitaminas lipossolúveis. O objetivo deste trabalho foi microencapsular vitamina D3, utilizando o método de spray chilling para a produção das micropartículas lipídicas sólidas (MLS). Para produção das MLS utilizou-se gordura vegetal com ponto de fusão em torno de 48 °C como carreador. Três tratamentos foram estabelecidos: sem aditivos (T1), com adição de 1% de cera de abelha (T2) e com 1% de lecitina de soja (T3). As micropartículas foram caracterizadas quanto à morfologia por microscopia eletrônica de varredura, tamanho médio por difração a laser, espectroscopia no infravermelho por transformada de Fourier (FTIR) e foi analisada a estabilidade da vitamina D3 durante o armazenamento a 10 e 25 °C, por meio de quantificações periódicas em cromatografia líquida de alta eficiência (CLAE). As micropartículas obtidas foram esféricas, semelhantes morfologicamente e com distribuição monocaudal de partículas. O tamanho médio das partículas variou em função dos seus ingredientes, sendo que as micropartículas produzidas apenas com vitamina e gordura foram menores em relação às demais (83,0% < 100 µm). A espectroscopia na região do infravermelho (FTIR) demonstrou que não ocorreu interação entre os ingredientes. A estabilidade da vitamina D3 encapsulada foi satisfatória ao longo de 65 dias com valores superiores a 87% para os três tratamentos e a temperatura apresentou influência na estabilidade. As MLS produzidas com cera apresentaram melhores resultados de estabilidade de vitamina D3 com valores de 90,18 ± 2,23 % após 65 dias de estocagem. Esses resultados são promissores e demostram a viabilidade da técnica de spray chilling na produção de MLS carregadas de vitamina D3, possibilitando uma futura aplicação em alimentos.

Differences in exposure to occupational health risks in Spanish and foreign-born workers in Spain (Itsal Project)

Migrant workers usually show higher rates of work-related health problems than natives. However, little information is available about their exposure to occupational risks. We describe self-reported working exposure in Spanish and foreign-born workers. A cross-sectional survey was conducted as part of the ITSAL Project. Data on sociodemographic and self-reported occupational exposure in 1,841 foreign-born and 509 Spanish workers were collected through face-to-face interviews. Prevalence and adjusted odds ratios-aOR- (by age, education, type of contract) were calculated. Foreign-born men in non-services sectors and those in manual occupations perceived exposure to occupational risks with lower prevalence than Spanish workers. Foreign-born women reported higher prevalence of exposure than Spanish female workers. By occupation, foreign-born female workers were more likely than Spanish workers to report working many hours/day (aOR2.68; 95 % CI 1.06–6.78) and exposure to extreme temperatures (aOR2.19; 95 % CI 1.10–4.38). Some groups of migrant workers may need increased protection regarding some occupational exposures.

Prevalence of exposure to occupational risks during preganancy in Spain

Objectives. We describe reported exposures to main categories of occupational agents and conditions in Spanish pregnant workers. Methods. Women were recruited at 12th week of pregnancy from main public gynaecological consults to be included in the INMA Spanish cohorts study (n = 2,058). Through personal interviews with structured questionnaires, information on working situation and working conditions during pregnancy was obtained. Results. Fifty percent of the women reported frequent exposure to physical load (standing, heavy lifting) and 45 % reported exposure to three or more indicators of job strain. Exposure to at least one physical agent (noise, vibrations, etc.) affected 25 % of the women. Exposure to chemicals was reported by 20 % of the women, mostly including solvents and cleaning products. Eight percent of the women worked at night shifts. Job strain was more prevalent in office workers and industrial operators. Industrial workers showed the highest prevalence of exposure to chemical and physical pollutants. Conclusions. Our data suggest that working conditions of pregnant women may need increased control in Spain.