The relationship between mental disorders and actual and desired subjective social status.

AIMS: Mental disorders are associated with lower subjective social status (SSS), but a more nuanced understanding of this relationship is needed. We examined the influence of disorder age of onset and recency on SSS and studied whether mental disorders are also associated with the discrepancy between actual and desired SSS. METHOD: Data are from the baseline and second wave of the Netherlands Mental Health Survey and Incidence Study-2 (NEMESIS-2). Mental disorders were assessed with the Composite International Diagnostic Interview (CIDI 3.0), while both actual and desired SSS were assessed with a ten-rung ladder. Linear regression was used to examine the association between mental disorders and SSS. RESULTS: Of 5303 participants, 2237 had a lifetime mental disorder at baseline. These participants reported significantly lower actual SSS (6.28) at follow-up than healthy participants (6.66, B = -0.38 [95% CI -0.48 to -0.27], p < 0.001) and a significantly greater actual-desired SSS discrepancy (1.14 v. 1.05 after controlling for actual SSS, B = 0.09 [0.01-0.17], p = 0.024). Lower age of onset of the first mental disorder was marginally significantly associated with lower actual SSS (B = 0.006 [0.000-0.012], p = 0.046). More recent disorders were also associated with lower actual SSS (B = 0.015 [0.005-0.026], p = 0.005), such that participants whose disorder remitted ⩾6 years before baseline were statistically indistinguishable from healthy participants. CONCLUSIONS: Lifetime mental disorders are associated with lower actual SSS and a slightly greater discrepancy between actual and desired SSS. However, people with mental disorders in (long-term) remission have a similar social status as healthy participants.

Spectroscopic (multi-energy) CT distinguishes iodine and barium contrast material in MICE.

OBJECTIVE: Spectral CT differs from dual-energy CT by using a conventional X-ray tube and a photon-counting detector. We wished to produce 3D spectroscopic images of mice that distinguished calcium, iodine and barium. METHODS: We developed a desktop spectral CT, dubbed MARS, based around the Medipix2 photon-counting energy-discriminating detector. The single conventional X-ray tube operated at constant voltage (75 kVp) and constant current (150 microA). We anaesthetised with ketamine six black mice (C57BL/6). We introduced iodinated contrast material and barium sulphate into the vascular system, alimentary tract and respiratory tract as we euthanised them. The mice were preserved in resin and imaged at four detector energy levels from 12 keV to 42 keV to include the K-edges of iodine (33.0 keV) and barium (37.4 keV). Principal component analysis was applied to reconstructed images to identify components with independent energy response, then displayed in 2D and 3D. RESULTS: Iodinated and barium contrast material was spectrally distinct from soft tissue and bone in all six mice. Calcium, iodine and barium were displayed as separate channels on 3D colour images at <55 microm isotropic voxels. CONCLUSION: Spectral CT distinguishes contrast agents with K-edges only 4 keV apart. Multi-contrast imaging and molecular CT are potential future applications.

Regulation of the Forkhead transcription factor AFX by Ral-dependent phosphorylation of threonines 447 and 451.

AFX is a Forkhead transcription factor that induces a G(1) cell cycle arrest via upregulation of the cell cycle inhibitor p27(Kip1). Previously we have shown that protein kinase B (PKB) phosphorylates AFX causing inhibition of AFX by nuclear exclusion. In addition, Ras, through the activation of the RalGEF-Ral pathway, induces phosphorylation of AFX. Here we show that the Ras-Ral pathway provokes phosphorylation of threonines 447 and 451 in the C terminus of AFX. A mutant protein in which both threonines are substituted for alanines (T447A/T451A) still responds to PKB-regulated nuclear-cytoplasmic shuttling, but transcriptional activity and consequent G(1) cell cycle arrest are greatly impaired. Furthermore, inhibition of the Ral signaling pathway abolishes both AFX-mediated transcription and regulation of p27(Kip1), while activation of Ral augments AFX activity. From these results we conclude that Ral-mediated phosphorylation of threonines 447 and 451 is required for proper activity of AFX-WT. Interestingly, the T447A/T451A mutation did not affect the induction of transcription and G(1) cell cycle arrest by the PKB-insensitive AFX-A3 mutant, suggesting that Ral-mediated phosphorylation plays a role in the regulation of AFX by PKB.

Ras-dependent regulation of c-Jun phosphorylation is mediated by the Ral guanine nucleotide exchange factor-Ral pathway.

The transcription factor c-Jun is critically involved in the regulation of proliferation and differentiation as well as cellular transformation induced by oncogenic Ras. The signal transduction pathways that couple Ras activation to c-Jun phosphorylation are still partially elusive. Here we show that an activated version of the Ras effector Rlf, a guanine nucleotide exchange factor (GEF) of the small GTPase Ral, can induce the phosphorylation of serines 63 and 73 of c-Jun. In addition, we show that growth factor-induced, Ras-mediated phosphorylation of c-Jun is abolished by inhibitory mutants of the RalGEF-Ral pathway. These results suggest that the RalGEF-Ral pathway plays a major role in Ras-dependent c-Jun phosphorylation. Ral-dependent regulation of c-Jun phosphorylation includes JNK, a still elusive JNKK, and possibly Src.

Direct control of the Forkhead transcription factor AFX by protein kinase B.

The phosphatidylinositol-3-OH-kinase (PI(3)K) effector protein kinase B regulates certain insulin-responsive genes, but the transcription factors regulated by protein kinase B have yet to be identified. Genetic analysis in Caenorhabditis elegans has shown that the Forkhead transcription factor daf-16 is regulated by a pathway consisting of insulin-receptor-like daf-2 and PI(3)K-like age-1. Here we show that protein kinase B phosphorylates AFX, a human orthologue of daf-16, both in vitro and in vivo. Inhibition of endogenous PI(3)K and protein kinase B activity prevents protein kinase B-dependent phosphorylation of AFX and reveals residual protein kinase B-independent phosphorylation that requires Ras signalling towards the Ral GTPase. In addition, phosphorylation of AFX by protein kinase B inhibits its transcriptional activity. Together, these results delineate a pathway for PI(3)K-dependent signalling to the nucleus.

The Rap1 GTPase functions as a regulator of morphogenesis in vivo.

The Ras-related Rap GTPases are highly conserved across diverse species but their normal biological function is not well understood. Initial studies in mammalian cells suggested a role for Rap as a Ras antagonist. More recent experiments indicate functions in calcium- and cAMP-mediated signaling and it has been proposed that protein kinase A-mediated phosphorylation activates Rap in vivo. We show that Ras1-mediated signaling pathways in Drosophila are not influenced by Rap1 levels, suggesting that Ras1 and Rap1 function via distinct pathways. Moreover, a mutation that abolishes the putative cAMP-dependent kinase phosphorylation site of Drosophila Rap1 can still rescue the Rap1 mutant phenotype. Our experiments show that Rap1 is not needed for cell proliferation and cell-fate specification but demonstrate a critical function for Rap1 in regulating normal morphogenesis in the eye disk, the ovary and the embryo. Rap1 mutations also disrupt cell migrations and cause abnormalities in cell shape. These findings indicate a role for Rap proteins as regulators of morphogenesis in vivo.

The actin binding domain of the epidermal growth factor receptor is required for EGF-stimulated tissue invasion.

NIH-3T3 fibroblasts expressing epidermal growth factor receptors (EGFRs) lacking the actin binding domain (ABD) were analyzed for their EGF-induced capacity to invade a bone marrow stromal cell (BMSC) monolayer. The fibroblasts display a reduction in the percentage of cytoskeleton-associated EGFRs. Furthermore, EGF-induced tyrosine kinase activity is unaffected by the mutation. Cells expressing the mutant EGFRs hardly invade a BMSC monolayer upon EGF stimulation in contrast to cells expressing wild-type EGFRs. Using the same cells no difference was observed in PDGF-induced invasion, which ligand was as potent in both cell types as EGF was in wild-type cells. Inhibition of both the phosphatidyl inositol-3-kinase (PI-3-K) and lipoxygenase pathways in wild-type cells mimicked the effect of the ABD deletion. Our results point to an important role for the ABD of the EGFR in EGF-induced tissue invasion.

Stimulation of gene induction and cell growth by the Ras effector Rlf.

Rlf is a ubiquitously expressed distinct relative of RalGDS that interacts with active Ras in vitro. We now demonstrate that Rlf, when co-expressed with Ras mutants, associates in vivo with RasV12 and the effector-domain mutant RasV12G37, but not with RasV12E38 or RasV12C40. Rlf exhibits guanine nucleotide exchange activity towards the small GTPase Ral and, importantly, Rlf-induced Ral activation is stimulated by active Ras. In addition, RasV12 and RasV12G37 synergize with Rlf in the transcriptional activation of the c-fos promoter. Rlf, when targeted to the plasma membrane using the Ras farnesyl attachment site (Rlf-CAAX), is constitutively active, inducing both Ral activation and c-fos promoter activity. Rlf-CAAX-induced gene expression is insensitive to dominant negative Ras and the MEK inhibitor PD98059, and involves activation of the serum response element. Furthermore, expression of Rlf-CAAX is sufficient to induce proliferation of NIH 3T3 cells under low-serum conditions. These data demonstrate that Rlf is an effector of Ras which functions as an exchange factor for Ral. Rlf mediates a distinct Ras-induced signalling pathway to gene induction. Finally, a constitutively active form of Rlf can stimulate transcriptional activation and cell growth.

Comparative study on metallothionein induction in whole testicular tissue and isolated Leydig cells.

The present study aimed at investigating whether previously reported interstrain differences of mice in testicular susceptibility to cadmium toxicity can be attributed to different metallothionein expression in the testes and other organs. Furthermore, purified Leydig cell systems were employed to identify a testicular compartment capable to respond specifically to exogenous cadmium by increased metallothionein expression. It was shown that species differences in testicular damage after cadmium administration can not be explained by differences in cadmium-induced stimulation of hepatic and renal metallothionein. In contrast to these organs, no metallothionein response to cadmium was observed in testicular homogenates. However, both freshly isolated and purified rat Leydig cells and a murine tumor Leydig cell line are not only able to synthesize metallothionein under basal conditions, but also respond to exogenous cadmium by increased synthesis of metallothionein (35S-cysteine incorporation) and an increased Mt-mRNA content. Extracts from freshly isolated and purified rat Leydig cells contained a cadmium-binding component which eluted in gel chromatography with the same properties as metallothionein. It was concluded that the use of purified cell systems is a valuable tool to investigate possible targets of cadmium toxicity.

Metallothionein induction by nonsteroidal antiinflammatory drugs.

In the present study we report on the effects of commonly used nonsteroidal antiinflammatory drugs on metallothionein (MT) and MT-I mRNA levels. A single dose of chloroquine (100 mg/kg), diclofenac (100 mg/kg), indomethacin (10 mg/kg), or piroxicam (100 mg/kg) was administered ip to C57B1 mice. After 18 h, MT levels were determined with a Cd-saturation radioassay. MT-I mRNA levels were measured by Northern Blot analyses using a probe containing the mouse MT-I gene. All drugs tested caused an increase in the MT content of the liver but not of the kidneys and lung. The lowest and highest effects were observed with chloroquine (8 times the control value) and diclofenac (18 times), respectively. In accordance with the stimulation of MT synthesis, increased accumulation of hepatic MT-I mRNA could be demonstrated. These results indicate that elevated MT levels may contribute to the effectiveness of nonsteroidal antiinflammatory drugs in the treatment of rheumatoid arthritis (RA).

Inhibition of hydroxyl-radical-generated DNA degradation by metallothionein.

The effect of metallothionein (MT) on hydroxyl-radical-induced DNA damage was investigated in an in-vitro study. The degradation of DNA as demonstrated by agarose gel electrophoresis was inhibited by MT in a concentration-dependent manner. The degradation of DNA was almost completely inhibited by a concentration of 13 microM MT, whereas a concentration of 10 mM glutathione was needed to achieve the same protective effect.

Effects of cadmium and lead on oxidative metabolism and phagocytosis by mouse peritoneal macrophages.

We investigated the effects of cadmium and lead on the oxidative metabolism of mouse peritoneal macrophages induced either by phagocytosis of zymosan particles or by membrane perturbation with phorbol myristate acetate (PMA) without phagocytosis. Oxidative metabolism activity was measured by chemiluminescence in the presence of lucigenin. Furthermore, cell viability was tested by dye exclusion test and phagocytic activity was measured by ingestion of latex particles. Within the first hour of exposure to the metals the effects of cadmium and lead in peritoneal macrophages were the same. The oxidative metabolism induced by PMA was enhanced by both metals, the zymosan-induced oxidative metabolism was reduced, markedly by cadmium and weakly by lead. Cell viability was not affected. After 20 h of exposure the effects of cadmium and lead were different. Cadmium did not alter either the oxidative metabolism induced by zymosan and PMA or phagocytosis of latex-particles. Lead concentration-dependently suppressed the oxidative metabolism induced by zymosan and PMA as well as phagocytosis of latex-particles. However, cadmium reduced cell viability concentration-dependently in contrast to lead that reduced cell viability only slightly.

Effect of heavy metals on cellular growth, metabolism and integrity of cultured Chinese hamster kidney cells.

The effects of CdCl2, Na2CrO4, NaAsO2 and NiSO4 on cultured Chinese hamster kidney cells were studied over a concentration range for 48 h. Release of lactate dehydrogenase, a parameter of cell viability, was not closely related to cell proliferation except for Na2CrO4. A better correlation was obtained between glucose consumption and lactate production, and cellular growth. When studying toxic effects of metals in cell-culture systems, metabolic parameters should be determined in addition to cell viability and cellular growth. The results indicate that Chinese hamster kidney cells in culture might be useful to study mechanisms of metal-induced toxicity.

Lipid peroxidation in isolated rat hepatocytes measured by ethane and n-pentane formation.

Isolated rat hepatocytes (1 X 10(7) cells/ml) were aerobically incubated in Eagle's Minimum Essential Medium which contained 2.0% albumin. As potential parameters of lipid peroxidation ethane and n-pentane formed were measured in samples obtained from the gas phase above the incubation mixture. 15-30 nmol ethane or n-pentane were produced by 10(7) hepatocytes within 90 min. Carbon tetrachloride (CCl4) or ADP-complexed ferrous ions stimulated ethane and n-pentane formation considerably, depending on the concentrations of the compounds. With CCl4 10(7) cells formed max 180 nmol ethane and 140 nmol n-pentane within 90 min incubation, whereas with Fe(II) max 130 nmol ethane and 220 nmol n-pentane could be detected. When n-pentane was added to the gas phase above the incubation mixture containing either medium or medium plus hepatocytes its amount decreased by 30% within the first 5 min of incubation. However, afterwards only minor amounts of n-pentane disappeared, even in the presence of hepatocytes. This indicates that n-pentane equilibrates with the cells suspension under the conditions used. Cell viability, as determined by the release of lactate dehydrogenase into the medium and by the uptake of trypan blue by the cells, and the recovery of the cells decreased only in presence of relatively high concentrations of CCl4, or Fe(II) respectively. However, a maximal effect on ethane and n-pentane formation was reached already with lower concentration.

Rat hepatic vinyl chloride metabolites induce gene conversion in the yeast strain D7RAD in vitro and in vivo.

We have tested the genetic activity of gaseous vinyl chloride in vitro and in vivo using the gene-conversion system (trp5-12/trp5-27 leads to TRP+) in the yeast strain D7RAD. To induce, in vitro, TRP+ convertants with 2.5% gaseous vinyl chloride, a rat-liver microsomal system for metabolic activation of the vinyl chloride and dividing yeast cells are required. Neither a deficiency in excision repair (rad3) nor in the error-prone repair pathway (rad6) increased the vinyl-chloride-induced conversion frequencies compared with the repair-competent D7RAD strain. When logarithmically growing cells of the D7RAD strain were injected intravenously into male Wistar rats which inhaled 1% vinyl chloride in air for 24 h, a significant enhancement of the TRP+ conversion frequencies was found compared with that in cells re-isolated from untreated rats. These results indicate that vinyl chloride metabolites from the metabolizing hepatocytes diffuse into yeast cells, which accumulate in the liver capillaries. This supports the hypothesis that the endothelial cells of the liver sinuses, which have hardly any metabolic activity, but give rise to vinyl-chloride-induced hemangiotheliomas (rare type of liver tumor), are transformed by diffusible metabolites of the procarcinogen vinyl chloride.

Ethane formation of isolated rat hepatocytes due to carbon tetrachloride.

Isolated rat hepatocytes incubated aerobically formed measurable amounts of ethane, a parameter for lipid peroxidation. This ethane formation increased several-fold due to carbon tetrachloride (CCl4) depending on its concentration. Cell damage as measured by trypan blue uptake and lactate dehydrogenase release poorly correlated with ethane formation. Ethane was not metabolized, whereas malondialdehyde (MDA), when added to isolated hepatocytes, decreased very rapidly. The results indicate that rather than MDA is a reliable parameter for lipid peroxidation occurring in isolated hepatocytes, and that a simple relationship between CCl4-induced lipid peroxidation and cell damage is not existing in isolated hepatocytes.

Ethane production of mouse peritoneal macrophages as indication for lipid peroxidation and the effect of heavy metals.

Mouse peritoneal macrophages cultivated for 24 h produced ethane which is a new index for lipid peroxidation reactions, whereas malondialdehyde (MDA), an alternative method, did not increase. This is explained by the metabolism of MDA by macrophages after addition of MDA, whereas the gaseous ethane added remained constant. This indicates that ethane rather than MDA is a reliable parameter for lipid peroxide formation by macrophages. Divalent heavy metal ions were examined for their effect on ethane formation and on cell viability. Whereas the amount of ethane formed by macrophages in presence of CdCl2 and MnCl2 remained fairly constant, PbCl2 and ZnCl2 resulted in decreased ethane production up to 60%. But with all these metals the viability decreased much more and did not correlate with the ethane formation. With some concentrations of FeCl2 an increased ethane formation was observed which was accompanied by decreased viability of the cells. The results are consistent with the view that heavy metals interfere with the oxygen radical reactions of macrophages, and that viability of macrophages is not closely related to these oxidative reactions.

[Analysis of the biological effect of city smog extract IV. Growth inhibition of kidney cell cultures (cercopithecus aethiops) under the influence of a city smog extract and its polyaromatic fractions (author's transl)]. / Untersuchung der biologischen Wirkung von atmosphärischen Feinstaubextrakten IV. Wachstumshemmung von Nierenzellkulturen (Cercopithecus aethiops) unter Einwirkung eines atmosphärischen Feinstaubextraktes und seiner Polyaromatenfraktionen.

The cell growth of exponentially growing kidney cell cultures of Cercopithecus aethiops was determined by estimation of protein content. The effect of city smog extracts and its polyaromatic fractions on cell growth was examined. Based on the benzo(a)pyren-content the crude extract of city smog exerted the strongest inhibition of cell growth, followed by non purified and purified fraction of polyaromates. The inhibition of cell growth was dose dependent. Results indicate, that for cell growth inhibition are of importance concentrations of toxic substances and exposition time.

[Analysis of the biological effect of city smog extract. III. Comparative investigations on the effect of city smog extracts on cell replication and DNA-synthesis of kidney cells in vitro from the primate Cercopithecus aethiops (author's transl)]. / Untersuchung der biologischen Wirkung von atmosphärischen Feinstaubextrakten. III. Vergleichende Untersuchungen über die Wirkung von Feinstaubextrakten auf die Zellvermehrung und DNS-synthese von Nierenzellen des Primaten Cercopithecus aethiops in vitro.

We analysed the cytotoxic effect of city smog extracts from a heavy industrialized area using kidney cell cultures from the primate Cercopithecus aethiops.--Using logarithmically growing cell cultures we determined cell replication and the rate of DNA-synthesis after incorporation of 3H-thymidine.--In presence of city smog extracts we found a dose dependent reduction of cell replication and of DNA-synthesis. In presence of high concentrations (BP-aquivalent 0.25-0.5 microgram/ml) of city smog extract we found no increase in cell number over a period of 72 h. Under the same conditions hardly any DNA-synthesis was detected.--In presence of middle and low concentrations of city smog extract a dose- and time-dependent increase in cell number and rate of DNA-synthesis was detected.

[Analysis of the biological effect of city smog extract. II. Effect of a city smog extract on cell growth and DNA synthesis of hamster kidney cells in vitro (author's transl)]. / Untersuchung der biologischen Wirkung von atmosphärischen Feinstaubextrakten. II. Einfluss eines atmosphärischen Feinstaubextraktes auf die Zellvermehrung und die DNS-Synthese von Hamsternierenzellen in vitro.

A city smog extract from an urban area inhibits the cell growth of hamster kidney cells in vitro. Parallel to an inhibition of cell multiplication a diminished rate of total DNA synthesis appeared. The number of cells in DNA synthesis is depressed in presence of city smog extract. These phenomena revealed a dose-response relationship. The biological effect of city smog extract is discussed.