Fos Synthesis Research Articles

Specific cell subclusters of dental pulp stem cells respond to distinct pathogens through the ROS pathway.

Microbial pathogens invade various human organs, including the oral cavity. Candida albicans (C.a) and Streptococcus mutans (S.m) served respectively as representative oral pathogenic fungi and bacteria to stimulate dental pulp stem cells (DPSCs) and to screen the DPSC subcluster that specifically responded to fungal infection. DPSCs were obtained from the impacted third molars of six healthy subjects. Then, cells were mixed and divided into three samples, two of which were stimulated with C.a and S.m, respectively; the third sample was exposed to cell medium only (Ctrl). Single-cell mRNA sequencing analysis of treated DPSCs was performed. DPSCs were composed of four major clusters of which one, DPSC.7, exhibited unique changes compared to those of other subclusters. The DPSC.7 cell percentage of the C.a sample was twice those of the Ctrl and S.m samples. DPSC.7 cells expressed genes associated with the response to reactive oxygen species (ROS) response. DPSC.7 subgroup cells established characteristic aggregation under the stimulation of different pathogens in UMAP. The MAPK/ERK1/2 and NF-κB pathways were up-regulated, DUSP1/5/6 expressions were suppressed, FOS synthesis was activated, the immune-related pathway was induced, and the levels of cytokines, including IL-6 and CCL2, were up-regulated in DPSC.7 cells when stimulated with C.a. Our study analyzed the cellular and molecular properties of DPSCs infected by oral fungi and bacteria with single-cell RNA sequencing. A subcluster of DPSCs responded specifically to infections with different pathogens, activating the MAPK and NF-κB pathways to induce immune responses via the ROS pathway. This suggests novel treatment strategies for fungal infections.

Simultaneous enzyme production, Levan-type FOS synthesis and sugar by-products elimination using a recombinant Pichia pastoris strain expressing a levansucrase-endolevanase fusion enzyme

BackgroundAlthough Levan-type fructooligosaccharides (L-FOS) have been shown to exhibit prebiotic properties, no efficient methods for their large-scale production have been proposed. One alternative relies on the simultaneous levan synthesis from sucrose, followed by endolevanase hydrolysis. For this purpose, several options have been described, particularly through the synthesis of the corresponding enzymes in recombinant Escherichia coli. Major drawbacks still consist in the requirement of GRAS microorganisms for enzyme production, but mainly, the elimination of glucose and fructose, the reaction by-products.ResultsThe expression of a fusion enzyme between Bacillus licheniformis endolevanase (LevB1) and B. subtilis levansucrase (SacB) in Pichia pastoris cultures, coupled with the simultaneous synthesis of L-FOS from sucrose and the elimination of the residual monosaccharides, in a single one-pot process was developed. The proof of concept at 250 mL flask-level, resulted in 8.62 g of monosaccharide-free L-FOS and 12.83 gDCW of biomass, after 3 successive sucrose additions (30 g in total), that is a 28.7% yield (w L-FOS/w sucrose) over a period of 288 h.At a 1.5 L bioreactor-level, growth considerably increased and, after 59 h and two sucrose additions, 72.9 g of monosaccharide-free L-FOS and 22.77 gDCW of biomass were obtained from a total of 160 g of sucrose fed, corresponding to a 45.5% yield (w L-FOS/w sucrose), 1.6 higher than the flask system. The L-FOS obtained at flask-level had a DP lower than 20 fructose units, while at bioreactor-level smaller oligosaccharides were obtained, with a DP lower than 10, as a consequence of the lower endolevanase activity in the flask-level.ConclusionWe demonstrate here in a novel system, that P. pastoris cultures can simultaneously be used as comprehensive system to produce the enzyme and the enzymatic L-FOS synthesis with growth sustained by sucrose by-products. This system may be now the center of an optimization strategy for an efficient production of glucose and fructose free L-FOS, to make them available for their application as prebiotics. Besides, P. pastoris biomass also constitutes an interesting source of unicellular protein.

Characterization of a novel endo-levanase from Azotobacter chroococcum DSM 2286 and its application for the production of prebiotic fructooligosaccharides

Prebiotics are known for their ability to modulate the composition of the human microbiome and mediate health-promoting benefits. Endo-levanases, which hydrolyze levan into short-chain FOS, could be used for the production of levan-based prebiotics. The novel endo-levanase (LevB2286) from Azotobacter chroococcum DSM 2286, combines an exceptionally high specific activity with advantageous hydrolytic properties. Starting from levan isolated from Timothy grass, LevB2286 produced FOS ranging from DP 2 – 8. In contrast to endo-levanases described in the literature, LevB2286 formed minor amounts of fructose and levanbiose, even with greatly extended incubation. The combined activity of LevB2286 and the levansucrase LevS1417 from Gluconobacter japonicus LMG 1417 led to a one-step synthesis of levan-type FOS from sucrose. 387.4 ± 17.3 g L−1 FOS were produced within 48 h by the production strategy based on crude cell extract of recombinant Escherichia coli expressing levS1417 and levB2286 simultaneously.

Enhancement of fructosyltransferase and fructooligosaccharides production by A. oryzae DIA-MF in Solid-State Fermentation using aguamiel as culture medium

The aim of this work was to improve the production of fructosyltransferase (FTase) by Solid-State Fermentation (SSF) using aguamiel (agave sap) as culture medium and Aspergillus oryzae DIA-MF as producer strain. SSF was carried out evaluating the following parameters: inoculum rate, incubation temperature, initial pH and packing density to determine the most significant factors through Box-Hunter and Hunter design. The significant factors were then further optimized using a Box-Behnken design and response surface methodology. The maximum FTase activity (1347U/L) was obtained at 32°C, using packing density of 0.7g/cm3. Inoculum rate and initial pH had no significant influence on the response. FOS synthesis applying the enzyme produced by A. oryzae DIA-MF was also studied using aguamiel as substrate.

Suckling-induced Fos activation and melanin-concentrating hormone immunoreactivity during late lactation

AimsMelanin-concentrating hormone (MCH) is implicated in the control of food intake, body weight regulation and energy homeostasis. Lactation is an important physiological model to study the hypothalamic integration of peripheral sensory signals, such as suckling stimuli and those related to energy balance. MCH can be detected in the medial preoptic area (MPOA), especially around the 19th day of lactation, when this hormone is described as displaying a peak synthesis followed by a decrease after weaning. The physiological significance of this phenomenon is unclear. Therefore, we aimed to investigate hypothalamic changes associated to sensory stimulation by the litter, in special its influence over MCH synthesis. Main methodsFemale Wistar rats (n=56) were euthanized everyday from lactation days 15–21, with or without suckling stimulus (WS and NS groups, respectively). MCH and Fos immunoreactivity were evaluated in the MPOA and lateral and incerto-hypothalamic areas (LHA and IHy). Key findingsSuckling stimulus induced Fos synthesis in all regions studied. An increase on the number of suckling-induced Fos-ir neurons could be detected in the LHA after the 18th day. Conversely, the amount of MCH decreased in the MPOA from days 15–21, independent of suckling stimulation. No colocalization between MCH and Fos could be detected in any region analyzed. SignificanceSuckling stimulus is capable of stimulating hypothalamic regions not linked to maternal behavior, possibly to mediate energy balance aspects of lactation. Although dams are hyperphagic before weaning, this behavioral change does not appear to be mediated by MCH.

Effects of lyophilization on catalytic properties of immobilized fructosyltransferase from Rhodotorula sp. LEB-V10

The extracellular fructosyltransferase (FTase) from Rhodotorula sp. LEB-V10 was immobilized on particles of niobium–graphite alloy and freeze dried (lyophilized), with and without additives. Twelve additives commonly applied as cryoprotectants were selected and evaluated both individually and in formulation; the biocatalyst was then studied according to its catalytic properties. Lyophilization with or without additives did not significantly affect the immobilized enzyme. After a period of 6 months, reductions in the initial enzymatic activity of about 7 and 4% were observed for the lyophilized enzyme when using 50 and 200mM sodium acetate buffers, respectively. CMC, sorbitol, inositol and trehalose as single additives (all at 2.5%, w/v) in 100mM sodium acetate buffer were capable to preserve the enzymatic activity after 6 months. However, formulations with more than one additive resulted in 36–14% less enzymatic activity after 6 months. After lyophilization, FOS synthesis features changed positively, by increasing the FOS yield from a non-lyophilizated yield of 58–68% with lyophilization. FOS composition changed as well, with 1F-fructofuranosyl-nystose (GF4) content increasing to 61% with lyophilization, which is 76 times higher than with the non-lyophilized enzyme.

Cytohesin Binder and Regulator Augments T Cell Receptor-induced Nuclear Factor of Activated T Cells·AP-1 Activation through Regulation of the JNK Pathway

Cytohesin binder and regulator (Cybr; also known as CYTIP, CASP, and PSCDBP) is a cytokine-induced gene preferentially expressed in hematopoietic tissues and in T helper 1 cells. Cybr protein associates with members of the cytohesin family, which are known ADP-ribosylation factors-GDP/GTP exchange factors, and its functions appear to regulate lymphocyte adhesion and cell-cell contact. Here we show that Cybr mRNA and protein levels are increased upon T cell receptor engagement. Cybr expression then influences T cell receptor-dependent signaling events, such as nuclear factor of activated T cells and AP-1 transcriptional activity. In addition, expression of Cybr results in increased T cell receptor-mediated activation of the Rho/Rac exchange factor Vav and of the JNK-p38 MAPK signaling pathway. The effects of Cybr on nuclear factor of activated T cells and AP-1 are dependent on MAPK activation, and enhanced activation of this cascade results in cooperation between the two transcription factors in the regulation of gene expression. These findings provide the first evidence that the adaptor protein Cybr not only regulates lymphocyte adhesion and cell-cell interaction but also contributes to the regulation of the signaling cascade and of the genetic program downstream of the T cell receptor.

Activation of feeding-related neural circuitry after unilateral injections of muscimol into the nucleus accumbens shell

Chemical inhibition of neurons in the nucleus accumbens shell (AcbSh) elicits intense, behaviorally specific, feeding in satiated rats. We have demonstrated previously that this treatment activates a number of brain regions, most significantly the lateral hypothalamus (LH). This activation could be elicited through a direct neural connection with the AcbSh or secondarily through changes in autonomic activity, stress, or circulating levels of orexigenic or satiety factors. In the present study, we used the immunohistochemical localization of Fos protein to map neuronal activation after unilateral muscimol injections into the AcbSh to determine whether AcbSh-mediated Fos expression remains lateralized in the circuit and whether secondary systemic changes in the rat can be excluded as primary factors in the activation of downstream component nuclei. Rats receiving only saline injections exhibited very little Fos immunoreactivity. In contrast, unilateral injections of muscimol into the AcbSh consistently increased Fos expression in several brain regions. Three distinct patterns of expression were observed. Fos synthesis in the LH was increased only on the side of the brain ipsilateral to the muscimol injection. Fos expression remained primarily ipsilateral to the injection site in the septohypothalamic, paraventricular hypothalamic (PVN), paratenial thalamic, and lateral habenular nuclei, and medial substantia nigra, but was increased bilaterally in the piriform cortex, supraoptic nucleus, central nucleus of the amygdala, and nucleus of the solitary tract. Smaller numbers of Fos-immunoreactive cells were seen unilaterally in the bed nucleus of the stria terminalis, medial ventral pallidum, arcuate nucleus, and ventral tegmental area and bilaterally in the supraoptic and tuberomammillary nuclei. The labeling in the LH, PVN, and other unilaterally labeled structures provides evidence that these brain regions are components of an AcbSh-mediated neural circuit and suggests that they may be involved in the expression of AcbSh-mediated feeding behavior.

Protein Kinase Cζ Phosphorylates Nuclear Factor of Activated T Cells and Regulates Its Transactivating Activity

Although several isoforms of protein kinase C (PKC) have been implicated in T lymphocyte activation events, little is known about their mode of action. To address the role of PKCzeta in T cell activation, we have generated Jurkat T cell transfectants expressing either the wild type (J-PKCzeta) or "kinase-dead" mutant (J-PKCzeta(mut)) versions of this protein. Expression of PKCzeta but not PKCzeta(mut) increased transcriptional activation mediated by the NF-kappaB or nuclear factor of activated T cells (NFAT). PKCzeta cooperates with calcium ionophore and with NFAT1 or NFAT2 proteins to enhance transcriptional activation of a NFAT reporter construct. However, neither NFAT nuclear translocation nor DNA binding were in J-PKCzeta cells. Our results show that PKCzeta enhanced transcriptional activity mediated by Gal4-NFAT1 fusion proteins containing the N-terminal transactivation domain of human NFAT1. Interestingly, PKCzeta synergizes with calcineurin to induce transcriptional activation driven by the NFAT1 transactivation domain. Co-precipitation experiments showed physical interaction between PKCzeta and NFAT1 or NFAT2 isoforms. Even more, PKCzeta was able to phosphorylate recombinant glutathione S-transferase-NFAT1 (1-385) protein. These data reveal a new role of PKCzeta in T cells through the control of NFAT function by modulating the activity of its transactivation domain.

Requirement for integration of phorbol 12-myristate 13-acetate and calcium pathways is preserved in the transactivation domain of NFAT1.

The transcription factor NFAT integrates signals from both calcium- and phorbol ester-stimulated signaling pathways. The calcium signal activates the calmodulin (CaM)-dependent phosphatase calcineurin, which dephosphorylates the regulatory domain of NFAT and promotes its nuclear import, while the phorbol ester signal results in synthesis and activation of Fos and Jun, transcription factors that bind cooperatively with the NFAT DNA-binding domain in the nucleus to mediate the transcription of many target genes. Here we show that transactivation by a GAL4 fusion protein containing the strong acidic N-terminal transactivation domain (TAD) of NFAT1 also requires both calcium and phorbol ester stimulation. The calcium requirement can be mimicked by coexpression of activated versions of two CaM-dependent enzymes, calcineurin and CaM kinase IV. Our data indicate that a 144-amino acid segment of NFAT1, containing the N-terminal TAD but lacking the DNA-binding and Fos/Jun interaction domains, resembles the full-length protein in requiring a combined input from two separate signaling pathways for optimal function in cells.

Long-lasting sensitization towards morphine in motoric and limbic areas as determined by c- fos expression in rat brain

Chronic application of morphine leads to the development of tolerance towards several of its effects, e.g., analgesia or respiratory depression. Simultaneously, however, sensitization arises which becomes apparent in behavioral tests as increased locomotion or increased self-application. A human correlate for the latter may be the increasing craving for opioids in addicts. To identify brain areas involved in these long-lasting processes, we studied the expression of the transcription factor c- fos by in situ hybridization in rat brain as a marker for changes in gene expression after single or repeated morphine applications in the animals. The only c- fos signal that exceeded background after a single dose of morphine (50 mg/kg) was a diffuse expression in the lateral septum. In contrast, repeated dosage twice daily for 10 days and ascending from 10 to 50 mg/kg resulted in a sharply delineated morphine-induced c- fos synthesis in the dorsomedial and lateral striatum, lateral septum, medial mammillary nuclei, anterior thalamus and, in part masked by a high background due to injection stress, in the cingulate cortex. Most of these areas belong to the limbic system or are closely associated with it. The c- fos response was inducible by morphine in pretreated animals for up to 8 weeks after finishing the repeated application scheme. Retrograde tracing studies revealed that the dorsomedial part of the striatum, which was strongly labeled with the c- fos probe, received inputs from limbic as well as from motoric parts of the thalamus and cortex. Therefore, the sensitization of morphine-induced c- fos expression in parts of the striatum seems to correlate with the locomotor effects of repeated morphine application, whereas the observed sensitization in several limbic brain areas might reflect emotional phenomena like increased self-administration in rats or drug craving in humans.

Light- and focus-dependent expression of the transcription factor ZENK in the chick retina.

Ocular growth and refraction are regulated by visual processing in the retina. We identified candidate regulatory neurons by immunocytochemistry for immediate-early gene products, ZENK (zif268, Egr-1) and Fos, after appropriate visual stimulation. ZENK synthesis was enhanced by conditions that suppress ocular elongation (plus defocus, termination of form deprivation) and suppressed by conditions that enhance ocular elongation (minus defocus, form deprivation), particularly in glucagon-containing amacrine cells. Fos synthesis was enhanced by termination of visual deprivation, but not by defocus and not in glucagon-containing amacrine cells. We conclude that glucagon-containing amacrine cells respond differentially to the sign of defocus and may mediate lens-induced changes in ocular growth and refraction.

Unilateral dopamine depletion paradoxically enhances amphetamine-induced Fos expression in basal ganglia output structures

The ability of amphetamine to induce expression of the immediate early gene protein, Fos, was examined by immunocytochemistry in animals with unilateral 6-hydroxydopamine lesions of the nigrostriatal bundle. Amphetamine induced Fos expression in the globus pallidus (GP) on the intact side of the brain, but this response was greatly attenuated on the dopamine-depleted side. In contrast, amphetamine induced little Fos expression in the entopeduncular nucleus (EPN) and the substantia nigra pars reticulata (SNpr) on the intact side of the brain, but resulted in pronounced expression in these structures on the lesioned side. These findings demonstrate that unilateral dopamine depletion results in a pathophysiological state in which some responses to amphetamine are attenuated while others are paradoxically potentiated. One explanation of these effects is that amphetamine may indirectly activate excitatory inputs to the SNpr and the EPN on both sides of the brain. On the intact side, these effects would be opposed by the simultaneous activation of inhibitory pathways arising in the striatum and the GP, with the result that little Fos expression would be seen. On the dopamine-depleted side, however, engagement of these inhibitory pathways would be attenuated and the unopposed effects of the excitatory inputs mobilized by amphetamine would result in exaggerated Fos synthesis.

Identification of kainate-preferring glutamate receptor subunit GluR7 mRNA and protein in the rat median eminence

In situ hybridization and immunohistochemistry were used to determine the presence of kainate-preferring glutamate receptor subunits GluR6 and GluR7 mRNA and protein in the median eminence of the rat. The results show that most tanycytes lining the ventral third ventricle and many astrocytes within the median eminence contain the GluR7 receptor subunit mRNA but not the GluR5 and GluR6 receptor subunit mRNA. Immunohistochemical stainings show that the GluR6/7 receptor protein was localized to tanycytic cell bodies, their basal processes and to many other astrocytes in different layers of the median eminence. The results suggest that glutamate can act directly on the glial cells in the median eminence by binding to the GluR7 subunit which may be important for the control of the secretion of releasing and inhibiting hormones from axon terminals in the external layer. In order to determine if these receptor subunits are functional, kainic acid was injected and c- fos expression monitored. Results show that kainic acid induced c- fos synthesis in most of these glial cells.

The daily pattern of Fos synthesis by hypothalamic pro-opiomelanocortin neurons is unaffected by adrenalectomy in the rat

At the onset of dark, a large population of rat mediobasal hypothalamic (MBH) pro-opiomelanocortin (POMC) neurons starts spontaneously expressing Fos-immunoreactivity (Fos-IR). Here we studied the effect of adrenalectomy upon this expression since circulating corticosteroids, which increase in the rat with the onset of behavioural wakening, are thought to modulate the basal expression of MBH POMC mRNA. Hence, groups of intact, adrenalectomised and sham-operated rats were sacrificed at times when Fos synthesis by POMC neurons is known to show either nadir (at light-offset) or peak (6 h after light-offset) values. Brains were processed for Fos- and/or POMC immunohistochemistry. This allowed us to show that, in all experimental groups, Fos-IR is hardly expressed in MBH POMC neurons at the onset of dark, whereas it is strongly induced 6h later. We concluded that such an induction is not triggered through the known evening rise of plasma corticosteroid levels.

Adrenalectomy does not affect the nocturnal peak of fos expression within hypothalamic pro‐opiomelanocortin neurons

Circulating glucocorticoid (GC) levels are thought to modulate the basal activity of pro-opiomelanocortin (POMC) neurons within the mediobasal hypothalamus (MBH) of the male rat. In a recent study we demonstrated that Fos-immunoreactivity (Fos-IR) was spontaneously induced throughout the dark phase of the light/dark cycle within a large population of these MBH neurons. Here, we have investigated the effect of adrenalectomy on the nocturnal expression of Fos protein within POMC neurons. To this aim, groups of intact (IN), adrenalectomized (ADX) and sham-operated (sham) rats were killed 7 days after surgery (or no surgery) at times when Fos-IR is known to show either nadir (at light offset) or peak (6 h after light offset) values within MBH POMC neurons. Brains were processed for Fos- and/or POMC-immunohistochemistry. The results showed that, at both times studied, 7-day adrenalectomy did not affect the number of POMC/Fos double-stained neurons within the MBH. The rostro-caudal pattern of distribution of such labeled neurons throughout the MBH of ADX rats was also similar to that of IN or sham rats. The present data demonstrate that the nocturnal induction of Fos within MBH POMC neurons is not controlled via the nychtemeral rhythm of secretion of the adrenal gland. Furthermore, this study shows that basal levels of circulating GC do not alter the nocturnal peak of Fos synthesis within POMC neurons.

Adrenalectomy does not affect the nocturnal peak of fos expression within hypothalamic pro-opiomelanocortin neurons

Circulating glucocorticoid (GC) levels are thought to modulate the basal activity of pro-opiomelanocortin (POMC) neurons within the mediobasal hypothalamus (MBH) of the male rat. In a recent study we demonstrated that Fos-immunoreactivity (Fos-IR) was spontaneously induced throughout the dark phase of the light/dark cycle within a large population of these MBH neurons. Here, we have investigated the effect of adrenalectomy on the nocturnal expression of Fos protein within POMC neurons. To this aim, groups of intact (IN), adrenalectomized (ADX) and sham-operated (sham) rats were killed 7 days after surgery (or no surgery) at times when Fos-IR is known to show either nadir (at light offset) or peak (6 h after light offset) values within MBH POMC neurons. Brains were processed for Fos- and/or POMC-immunohistochemistry. The results showed that, at both times studied, 7-day adrenalectomy did not affect the number of POMC/Fos double-stained neurons within the MBH. The rostro-caudal pattern of distribution of such labeled neurons throughout the MBH of ADX rats was also similar to that of IN or sham rats. The present data demonstrate that the nocturnal induction of Fos within MBH POMC neurons is not controlled via the nychtemeral rhythm of secretion of the adrenal gland. Furthermore, this study shows that basal levels of circulating GC do not alter the nocturnal peak of Fos synthesis within POMC neurons.

Regulation of urokinase-type plasminogen activator expression by an ERK1-dependent signaling pathway in a squamous cell carcinoma cell line.

The urokinase-type plasminogen activator contributes to tissue remodeling by controlling the synthesis of the extracellular matrix-degrading plasmin. We undertook a study to determine the role of the extracellular signal-regulated kinases (ERKs) in the regulation of urokinase-type plasminogen activator expression in a squamous cell carcinoma cell line (UM-SCC-1) that contains a transcriptionally activated urokinase-type plasminogen activator gene. Transient transfection studies using a CAT reporter driven by the urokinase-type plasminogen activator promoter, which had progressive 5' deletions or which had been point-mutated, indicated the requirement of binding sites for AP-1 (-1967) and PEA3 (-1973) for its maximal activation. Expression of a mutant jun protein, which lacks the transactivation domain, caused a dose-dependent repression of a CAT reporter driven by either the urokinase-type plasminogen activator promoter or three tandem AP-1 repeats upstream of a thymidine kinase minimal promoter indicating the importance of AP-1-binding transcription factor(s) in the regulation of urokinase-type plasminogen activator synthesis. Mobility shift assays with UM-SCC-1 nuclear extract revealed binding of fos and junD proteins to an oligonucleotide spanning the AP-1 site at -1967. In-gel kinase assays indicated the constitutive activation of ERK1, which regulates fos synthesis via phosphorylation of p62TCF, but not ERK2, in UM-SCC-1 cells. Moreover, the expression of a dominant-negative ERK1, but not ERK2, repressed urokinase-type plasminogen activator promoter activity. Similarly, interfering with the function of the c-raf serine-threonine kinase, which lies upstream of ERK1, by the expression of a kinase-inactive c-raf repressed the activity of a CAT reporter driven by either the urokinase-type plasminogen activator promotor or tandem AP-1 repeats. These data suggest that urokinase-type plasminogen activator expression in UM-SCC-1 cells is regulated partly by an ERK1, but not ERK2, -dependent signaling pathway.

Interleukin 1-induced Fos and Jun do not regulate inducible nitric oxide synthase in rat islets of Langerhans and RINm5F cells.

Recent evidence indicates that nitric oxide (NO) produced after expression of inducible NO synthase (iNOS) mediates cytokine-induced inhibition of insulin secretion by pancreatic islets. The current studies were designed to characterize the involvement of immediate-early response genes, c-fos and c-jun, in interleukin 1 (IL-1)-induced expression of iNOS. iNOS messenger RNA (mRNA) expression by both rat islets and RINm5F cells was time dependent, with maximal expression observed after an approximately 3- to 6-h exposure to IL-1. IL-1 also stimulated rapid and transient expression of c-fos and c-jun by both rat islets and RINm5F cells, with maximal mRNA accumulation detected 30-60 min after IL-1 treatment. IL-1-induced protein synthesis of Fos and Jun was observed as early as 30 min, peaked between 3-5 h, and decreased by 8 h after IL-1 treatment. Temporal correlation of Fos and Jun expression and iNOS gene induction suggested that Fos and Jun might regulate iNOS gene transcription by rodent pancreatic beta-cells. The present study, however, indicates that IL-1 induced expression of Fos and Jun does not seem to participate in the regulation of iNOS and mRNA expression, because: 1) cycloheximide (1 microM) completely inhibited Fos expression but had no inhibitory effect on iNOS mRNA levels; and 2) tyrosine kinase inhibitors genistein and herbimycin A completely inhibited IL-1 induced iNOS expression but did not block c-fos and c-jun expression. These results indicate that two separate signaling pathways may exist for induction of c-fos and c- jun and iNOS genes and that de novo synthesis of Fos and Jun does not participate in the regulation of iNOS gene expression.

The cell-coded polypeptide U90 increased by herpes simplex virus type 2 infection induces Fos and DNA synthesis.

Lytic infection with herpes simplex virus type 2 (HSV-2) induces synthesis of a cell-coded protein of molecular mass 90 kDa, termed U90. U90, whose expression is specific in tumour cells is, in addition, excreted from he cell. To determine if the function of U90 could be advantageous for the virus the protein was purified from the medium of HSV-2 infected cells, shown to be similar to the internalized form and its effect on cell growth studied. Addition of U90 to quiescent fibroblast cells stimulated teh expression of Fos, the product of the cellular transcription factor c-fos and increased the incorporation of [3H]thymidine into DNA, factors which may facilitate HSV-2 replication. However, U90 might also induce abnormal cells such as those in precancerous lesions to proliferate.