Mechanisms of synaptic depression triggered by metabotropic glutamate receptors.

Glutamate, by activation of metabotropic receptors (mGluRs), can lead to a reduction of synaptic efficacy at many synapses. These forms of synaptic plasticity are referred to as long-term depression (mGluR-LTD). We will distinguish between mGluR-LTD induced by pre- or postsynaptic receptors and mGluR-LTD induced by the locus of the expression mechanism of the synaptic depression. We will also review recent evidence that mGluR-mediated responses themselves are subject to depression, which may constitute a form of metaplasticity.

Effects of streptozotocin-diabetes on the hippocampal NMDA receptor complex in rats.

In animal models of diabetes mellitus, such as the streptozotocin-diabetic rat (STZ-rat), spatial learning impairments develop in parallel with a reduced expression of long-term potentiation (LTP) and enhanced expression of long-term depression (LTD) in the hippocampus. This study examined the time course of the effects of STZ-diabetes and insulin treatment on the hippocampal post-synaptic glutamate N-methyl-D-aspartate (NMDA) receptor complex and other key proteins regulating hippocampal synaptic transmission in the post-synaptic density (PSD) fraction. In addition, the functional properties of the NMDA-receptor complex were examined. One month of STZ-diabetes did not affect the NMDA receptor complex. In contrast, 4 months after induction of diabetes NR2B subunit immunoreactivity, CaMKII and Tyr-dependent phosphorylation of the NR2A/B subunits of the NMDA receptor were reduced and alphaCaMKII autophosphorylation and its association to the NMDA receptor complex were impaired in STZ-rats compared with age-matched controls. Likewise, NMDA currents in hippocampal pyramidal neurones measured by intracellular recording were reduced in STZ-rats. Insulin treatment prevented the reduction in kinase activities, NR2B expression levels, CaMKII-NMDA receptor association and NMDA currents. These findings strengthen the hypothesis that altered post-synaptic glutamatergic transmission is related to deficits in learning and plasticity in this animal model.

Cyclin-dependent kinase 2 nucleocytoplasmic translocation is regulated by extracellular regulated kinase.

Activation of cyclin-dependent kinase 2 (CDK2)-cyclin E in the late G(1) phase of the cell cycle is important for transit into S phase. In Chinese hamster embryonic fibroblasts (IIC9) phosphatidylinositol 3-kinase and ERK regulate alpha-thrombin-induced G(1) transit by their effects on cyclin D1 protein accumulation (Phillips-Mason, P. J., Raben, D. M., and Baldassare, J. J. (2000) J. Biol. Chem. 275, 18046-18053). Here, we show that ERK also affects CDK2-cyclin E activation by regulating the subcellular localization of CDK2. Ectopic expression of cyclin E rescues the inhibition of alpha-thrombin-induced activation of CDK2-cyclin E and transit into S phase brought about by treatment of IIC9 cells with LY29004, a selective inhibitor of mitogen stimulation of phosphatidylinositol 3-kinase activity. However, cyclin E expression is ineffectual in rescuing these effects when ERK activation is blocked by treatment with PD98059, a selective inhibitor of MEK activation of ERK. Investigation into the mechanistic reasons for this difference found the following. 1) Although treatment with LY29004 inhibits alpha-thrombin-stimulated nuclear localization, ectopic expression of cyclin E rescues CDK2 translocation. 2) In contrast to treatment with LY29004, ectopic expression of cyclin E fails to restore alpha-thrombin-stimulated nuclear CDK2 translocation in IIC9 cells treated with PD98059. 3) CDK2-cyclin E complexes are not affected by treatment with either inhibitor. These data indicate that, in addition to its effects on cyclin D1 expression, ERK activity is an important controller of the translocation of CDK2 into the nucleus where it is activated.

Protein kinase C activation modulates alpha-calmodulin kinase II binding to NR2A subunit of N-methyl-D-aspartate receptor complex.

The N-methyl-d-aspartate (NMDA) receptor subunits NR2 possess extended intracellular C-terminal domains by which they can directly interact with a large number of postsynaptic density (PSD) proteins involved in synaptic clustering and signaling. We have previously shown that PSD-associated alpha-calmodulin kinase II (alphaCaMKII) binds with high affinity to the C-terminal domain of the NR2A subunit. Here, we show that residues 1412-1419 of the cytosolic tail of NR2A are critical for alphaCaMKII binding, and we identify, by site directed mutagenesis, PKC-dependent phosphorylation of NR2A(Ser(1416)) as a key mechanism in inhibiting alphaCaMKII-binding and promoting dissociation of alphaCaMKII.NR2A complex. In addition, we show that stimulation of PKC activity in hippocampal slices either with phorbol esters or with the mGluRs specific agonist trans-1-amino-1,3- cyclopentanedicarboxylic acid (t-ACPD) decreases alphaCaMKII binding to NMDA receptor complex. Thus, our data provide clues on understanding the molecular basis of a direct cross-talk between alphaCaMKII and PKC pathways in the postsynaptic compartment.

Evidence for the involvement of two different MHC class II regions in susceptibility or protection in allergic bronchopulmonary aspergillosis.

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) is a disease with uncertain pathology. Studies have suggested a pathogenic role for T(H)2 cells. Previously, we demonstrated, in a small group of patients, that T(H)2 reactivity to a major Aspergillus fumigatus antigen was restricted by HLA-DR2 or HLA-DR5 alleles. OBJECTIVES: We sought to confirm whether susceptibility to ABPA is exclusively associated with HLA-DR locus and to investigate the involvement of HLA-DQ genes in the development of ABPA. METHODS: Genomic DNA was extracted from patients with ABPA, patients without ABPA but with positive A fumigatus skin test responses and asthma or cystic fibrosis, and healthy control subjects. HLA-DR and HLA-DQ genes were detected by using low-resolution typing; high-resolution typing was done only on HLA-DR2- and HLA-DR5-positive individuals by using sequence-specific primers (PCR-SSP). RESULTS: A significantly higher frequency of HLA-DR2 was observed in patients with ABPA versus those without ABPA (corrected P <.01) or healthy control subjects (corrected P <.01). Genotype analysis revealed that susceptibility to ABPA is associated with HLA-DR2 alleles DRB1*1503 and DRB1*1501 and, to a lesser extent, with the HLA-DR5 allele DRB1*1104. The presence of DR4 or DR7 alleles in non-DR2/5 patients with ABPA suggests that these alleles may also be contributing factors in this disease. Another striking observation was the significantly high frequency of HLA-DQ2 in patients without ABPA (67. 4%) compared with patients with ABPA (20.5%) and normal control subjects (37.7%), suggesting that these alleles may confer protection in the population without ABPA. CONCLUSION: These genetic studies suggest that HLA-DR molecules DR2, DR5, and possibly DR4 or DR7 contribute to susceptibility while HLA-DQ2 contributes to resistance and that a combination of these genetic elements determines the outcome of ABPA in patients with cystic fibrosis and asthma.

Dry adsorbed emulsion: 1. Characterization of an intricate physicochemical structure.

A recent solid pharmaceutical form called "Dry Adsorbed Emulsion" (DAE) was characterized in morphological and structural fields. A DAE is an intricate system initiated by a water-in-oil emulsion including the active drug (i.e., theophylline). Each emulsion phase is adsorbed on pulverulent adsorbents with a suitable polarity (silica) to obtain a free-flowing powder with nonporous particles of size from 125 to 710 microm, with small specific surface area and a spherical shape. Different methods, such as scanning electron microscopy combined with chemical microanalysis, dying tests, and electron spin resonance studies, allow the formulator to follow the behavior of DAE aqueous and oily phases during the manufacturing process and then to set up a structural model for DAE particles. These DAE particles appear to be made up of a random pack of hydrophilic and hydrophobic particles, containing a liquid phase adsorbed on silica by weak bonds.

Brief hypoxia differentially regulates LPS-induced IL-1beta and TNF-alpha gene transcription in RAW 264.7 cells.

Episodes of tissue hypoxia and reoxygenation frequently occur during gram-negative bacteremia that progresses to septic shock. However, few studies have evaluated modulation by hypoxia and reoxygenation of the proinflammatory cytokine gene expression that is normally induced by gram-negative bacteremia or endotoxemia. In buffer-perfused organs, hypoxia downregulates Escherichia coli-induced expression of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta in the liver but upregulates these cytokines in the lungs. To identify molecular mechanisms underlying these events, we investigated the effects of brief (1.5-h) hypoxia on TNF-alpha and IL-1beta expression in cultured RAW 264.7 cells during their continuous exposure to lipopolysaccharide (LPS) endotoxin derived from E. coli (serotype 055:B5) for up to 24 h. IL-1beta and TNF-alpha concentrations in cell lysates and culture supernatants were measured by ELISA, and steady-state mRNA was measured by Northern analysis. LPS-induced IL-1beta synthesis was downregulated by hypoxia at both the protein and mRNA levels despite no change in cellular redox status as measured by levels of GSH. In contrast, LPS-induced TNF-alpha production was unaffected by hypoxia as assessed by cell lysate mRNA and lysate and supernatant protein levels. Nuclear runoff analysis showed that downregulation of IL-1beta gene expression by hypoxia occurred transcriptionally. Allopurinol or catalase treatment did not alter modulation of LPS-induced IL-1beta expression by hypoxia, suggesting that this suppression was not caused by reactive oxygen species. Cycloheximide pretreatment suggested that hypoxia-induced downregulation of IL-1beta expression did not require de novo protein synthesis.

The role of p38 mitogen-activated protein kinase in IL-1 beta transcription.

Several reports have shown that bicyclic imidazoles, specific inhibitors of the p38 mitogen-activated protein kinase (MAPK), block cytokine synthesis at the translational level. In this study, we examined the role of p38 MAPK in the regulation of the IL-1beta cytokine gene in monocytic cell lines using the bicyclic imidazole SB203580. Addition of SB203580 30 min before stimulation of monocytes with LPS inhibited IL-1beta protein and steady state message in a dose-dependent manner in both RAW264.7 and J774 cell lines. The loss of IL-1beta message was due mainly to inhibition of transcription, since nuclear run-off analysis showed an approximately 80% decrease in specific IL-1 RNA synthesis. In contrast, SB203580 had no effect on the synthesis of TNF-alpha message. LPS-stimulated p38 MAPK activity in the RAW264.7 cells was blocked by SB203580, as measured by the inhibition of MAPKAP2 kinase activity, a downstream target of the p38 MAPK. CCAATT/enhancer binding protein (C/EBP)/NFIL-6-driven chloramphenicol acetyltransferase (CAT) reporter activity was sensitive to SB203580, indicating that C/EBP/NFIL-6 transcription factor(s) are also targets of p38 MAPK. In contrast, transfected CAT constructs containing NF-kappaB elements were only partially inhibited (approximately 35%) at the highest concentration of SB203580 after LPS stimulation. As measured by EMSA, LPS-stimulated NF-kappaB activation was not affected by SB203580. Overall, the results demonstrate, for the first time, a role for p38 MAPK in IL-1beta transcription by acting through C/EBP/NFIL-6 transcription factors.

RhoA stimulates p27(Kip) degradation through its regulation of cyclin E/CDK2 activity.

RhoA has been identified as an important regulator of cell proliferation. We recently showed that the Ras/RhoA pathway regulates the degradation of p27(Kip) and the progression of Chinese hamster embryo fibroblasts (IIC9 cells) through G1 into S phase (Weber, J. D., Hu, W., Jefcoat, S. C., Raben, D. M., and Baldassare, J. J. (1997) J. Biol. Chem. 272, 32966-32971). In this report, we have demonstrated that, in IIC9 cells, RhoA regulates cyclin E/CDK2 activity, which is required for p27(Kip) degradation. As previously shown in several fibroblasts cell lines, expression of dominant-negative CDK2 in IIC9 cells blocked serum-induced cyclin E/CDK2 activity and p27(Kip) degradation. In the absence of serum, expression of constitutively active RhoA(63) resulted in significant stimulation of cyclin E/CDK2 activity and degradation of p27(Kip). Cotransfection of dominant-negative CDK2 and RhoA(63) inhibited RhoA(63)-induced cyclin E/CDK2 activity and p27(Kip) degradation. In addition, expression of dominant-negative RhoA blocked serum-induced cyclin E/CDK2 activity and p27(Kip) degradation. Finally, expression of catalytically active cyclin E/CDK2 rescued the effect of expression of dominant-negative RhoA. Taken together, these data show that RhoA regulates p27(Kip) degradation through its regulation of cyclin E/CDK2 activity.

Effect of environmental estrogens on IL-1beta promoter activity in a macrophage cell line.

Environmental estrogens or estrogen disrupters have recently received a great deal of attention because of their potential health impact on reproductive tissues. Few, if any, studies have been made on the impact of these compounds on the immune system. We sought to determine the activities of various environmental estrogens on the modulation of the interleukin-1beta (IL-1beta) gene in a model monocytic cell line, hER + IL-1beta-CAT+. This cell line stably transfected with the human estrogen receptor, and an IL-1beta promoter construct fused to the CAT reporter gene allows us to monitor the effect of estrogenic compounds on IL-1beta promoter activity. 17beta-estradiol (E2) markedly enhanced lipopolysaccharide- (LPS) induced IL-1beta promoter-driven CAT activity in a dose-dependent manner. The mycotoxins alpha-zearalenol and zearalenone both exhibited full agonist activity, but at lower potencies, with EC50 values of 1.8 and 54 nM, respectively, compared with E2 at 0.5 nM. In addition, genistein was a very low-potency agonist, having an EC50 of 1.5 microM. Similar to the E2 response, the slope factors for alpha-zearalenol, zearalenone, and genistein were close to 3.0, suggesting positive cooperativity in the estrogenic response. The activity of the mycotoxins appeared to be mediated through the estrogen receptor, since both the antiestrogens H1285 and ICI 182,780 effectively inhibited their agonist activity in a dose-dependent manner. Representative environmental estrogenic compounds both from plant and industrial sources were also tested. Unlike the mycoestrogens, none of the compounds, with the exception of genistein, synergized with LPS to enhance IL-1beta promoter activity. When tested for antiestrogenic activity, the industrial compound 4-octylphenol was able to antagonize the response to E2; however, the response was three orders of magnitude less potent than H 1285. Naringenin, a plant flavonoid, showed little or no ability to antagonize the response to E2. Overall, the results show that some environmental estrogens that display agonist activity in reproductive tissue also have an effect on IL-1 gene expression in hemopoietic-derived tissue.

Effect of estrogens on IL-1beta promoter activity.

It is well documented that steroid hormones modulate cytokine gene expression. In some tissues estrogens are known to suppress cytokine production while in other tissue types, cytokine expression is enhanced by the hormone. This study was conducted to investigate the regulatory mechanisms which underlie the modulation of the interleukin-1beta (IL-1beta) gene at the transcription level. To accomplish this, the macrophage cell line RAW264.7, which appeared insensitive to 17beta-estradiol (E2) treatment, was stably transfected with the human estrogen receptor (ER) and an IL-1beta promoter-CAT reporter construct. E2 markedly enhanced LPS-induced IL-1beta promoter-driven CAT activity in an E2 dose dependent manner. This responsiveness was estrogen specific since no synergism was observed between LPS and the sex steroids testosterone or progesterone while the estrogen analogue 17alpha-estradiol stimulated only at 10 to 100 times the amount required for 17beta-E2. Several antiestrogens, H1285, ICI 182 780, and tamoxifen inhibited the estrogen stimulated enhancement of IL-1beta promoter activity in a dose-dependent manner, indicating that this effect was indeed mediated through the ER in a ligand dependent manner. The estrogenic effect appeared to be indirect and time dependent since the addition of E2 was required hours prior to LPS stimulation; addition of E2 and LPS at the same time resulted in a greatly reduced estrogenic effect. The estrogen metabolites 17-epiestriol and 16-keto-17beta-E2 displayed an estrogenic response virtually indistinguishable from E2. 4-Hydroxyestradiol displayed activity only at 100-fold the concentration of E2 while 2-hydroxyestrone showed no activity at any of the concentrations tested. Overall the results demonstrate that E2 and some metabolites of E2 synergize with LPS to markedly enhance IL-1beta promoter activity through ER mediated processes.

The association of HLA-DR alleles and T cell activation with allergic bronchopulmonary aspergillosis.

Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity lung disease caused by the mold Aspergillus fumigatus. We previously reported that the majority of T cell clones (TCC) isolated from three ABPA patients, and specific for a dominant Ag of A. fumigatus, Asp f 1, were IL-4-producing CD4+ Th2 cells capable of responding to Ag in association with the HLA-DR subtypes DRB1*1501, *1503, and *1601 for HLA-DR2, and DRB1*1101, *1104, and *1202 for HLA-DR5. In the present study we extended the previous findings to determine whether the observed restriction with the HLA-DR2/5 subtypes held importance in a larger patient population. Serotyping revealed that 16 of 18 ABPA patients were either HLA-DR2, HLA-DR5, or both. Compared with a normal control population, the frequencies of HLA-DR2 (50 vs 22.3%) and HLA-DR5 (44.4 vs 19.8%) were significantly increased in these ABPA patients. Genotype analyses of an additional 15 patients identified the same HLA-DR subtypes previously shown functional for Asp f 1 Ag presentation. The relative avidities of Asp f 1 peptides for the purified HLA-DR subtypes, DRB1*1501 (functional) and DRB1*1502 (nonfunctional), were examined to determine whether differential binding to the HLA-DR subtypes explains successful Ag presentation. Similar low binding avidities were detected for both HLA-DR subtypes, indicating that the functionality cannot be simply explained by differences in binding affinities. Thus, the limited number and their role in Ag presentation emphasizes the possibility that the six identified HLA-DR subtypes are important in the pathophysiology of ABPA.

Characterization and localization of epidermal growth factor receptors in human developing tooth.

In this study the characterization and localization of Epidermal Growth Factor (EGF) receptor in human jaws, from fetuses ranging in age from 9 to 12 weeks is reported for the first time. Binding of [125I]-EGF to membranes obtained from three separate pools of fetal jaws was specific and time- and temperature-dependent. Analysis of the binding data revealed the presence of a single class of binding site with high affinity (Kd, 9.2 x 10(-10) mol/L) and mean binding capacity of 128 fmoles/mg protein. Immunohistochemical study demonstrated the presence of EGF receptors in the early developmental stages of human tooth. In the bud stage, the positivity was localized in the epithelial cells. In the cap stage, EGF receptors was present in the outer and inner enamel cells, in some cells of the stellate reticulum and in the mesenchymal papilla and follicle cells. In the bell stage, positivity for EGF receptors was present in the outer enamel epithelium, in some cells of the stellate reticulum and in the mesenchymal cells of the follicle and papilla. The presence of EGF receptors in the proliferative stages in both epithelial and mesenchymal cells suggests that EGF is involved in the early developmental stages of the human tooth germ.

T cell subsets, epitope mapping, and HLA-restriction in patients with allergic bronchopulmonary aspergillosis.

Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity lung disease characterized by Aspergillus fumigatus (Af) colonization, IgE and IgG anti-Af antibodies, pulmonary infiltrates, bronchiectasis, and pulmonary fibrosis. Little is known regarding T cell responses and their role in the pathogenesis of ABPA. To examine T cell reactivity to Af antigens, T cell clones (TCC) specific to the Asp f 1 antigen, an 18-kD protein of Af, were established from the peripheral blood of three ABPA patients. The majority of TCC isolated from ABPA patients, and specific for the Asp f 1 allergen of Af, are IL-4 producing CD4+ cells of the Th2 phenotype. Further analysis in this study revealed that the majority of TCC reacted to mainly two epitopes of Asp f 1, while the remaining TCC reacted to three additional "minor" epitopes. Blocking studies using monoclonal antibodies specific for class II HLA-D region gene products showed that most TCC, 19/21, were restricted by HLA-DR molecules, and the remaining two clones by HLA-DP molecules. The use of a panel of HLA-matched and mismatched EBV-transformed B cells as antigen presenting cells revealed that the HLA-DR restriction was mediated exclusively by either the HLA-DR2 or HLA-DR5 alleles. Genotyping of DRB1 gene products showed that class II presentation for most clones was not restricted to a single allele, representing DRB1 gene products of either HLA-DR2 or DR5. These studies offer insight into the cellular and molecular determinants which contribute to the immunopathophysiology of ABPA.

A novel cis-acting element required for lipopolysaccharide-induced transcription of the murine interleukin-1 beta gene.

Regulatory elements important for transcription of the murine interleukin-1 beta (IL-1 beta) gene lie within a DNase I-hypersensitive region located > 2,000 bp upstream from the transcription start site. We have identified within this region a novel positive regulatory element that is required for activation of an IL-1 beta promoter-chloramphenicol acetyltransferase (CAT) fusion gene in the murine macrophage line RAW264.7. Electrophoretic mobility shift analysis of the 3' portion (-2315 to -2106) of the hypersensitive region revealed at least two nuclear factor binding sites, one of which is located between positions -2285 and -2256. Competitive inhibition studies localized the binding site to a 15-bp sequence between -2285 and -2271. Nuclear factor binding was lost by mutation of the 6-bp sequence from -2280 to -2275. The specific retarded complex formed with RAW264.7 nuclear extract was not detected under similar conditions with nuclear extracts from RLM-11, a murine T-cell line which does not express IL-1 beta RNA. Mutation of the 6-bp sequence (-2280 to -2275) in the chimeric IL-1 beta promoter -4093 +I CAT plasmid virtually eliminated the activation of this reporter gene by lipopolysaccharide (LPS) in transfected RAW264.7 cells. Multimerization of the 15-bp sequence containing the core wild-type 6-bp sequence 5' of minimal homologous or heterologous promoters in CAT reporter plasmids resulted in significant enhancement of CAT expression compared with parallel constructs containing the mutant 6-bp core sequence. This element was LPS independent and position and orientation dependent. The multimerized 15-bp sequence did not enhance expression in RLM-11 cells. Methylation interference revealed contact residues from -2281 to -2271, CCAAAAAGGAA. Because a search of the NIH TFD data bank with the 11-bp binding site sequence found no homology to known nuclear factor binding sites, we have designated this sequence the IL1 beta -upstream nuclear factor 1 (IL1 beta -UNF1) target. UV cross-linking and sodium dodecyl sulfate-polyacrylamide electrophoresis identified an IL1 beta -UNF1-specific binding factor approximately 85 to 90 kDa in size.

Upstream NFIL-6-like site located within a DNase I hypersensitivity region mediates LPS-induced transcription of the murine interleukin-1 beta gene.

To define the cis-acting elements that regulate LPS-stimulated IL-1 beta gene transcription, we analyzed the murine IL-1 beta gene by digestion with DNase I. At least two hypersensitive sites were located between 2200 and 2600 bp upstream of the transcription start site in mononuclear phagocytes, but not in an IL-1 nonproducing immature T cell line. Specific DNA sequences required for LPS induction of IL-1 beta gene expression were identified within the DNase I hypersensitive (DH) region using transfection of reporter constructs that contained portions of the IL-1 beta 5'-flanking region. Two specific DNA sequences were targets for nuclear factor binding as assessed with use of electrophoretic mobility shift analysis (EMSA). One site contained a consensus sequence for NFIL-6 binding. Base substitutions within this NFIL-6 site resulted in virtual elimination of LPS-induced IL-1 beta gene transcription. Introduction of multimers of the NFIL-6-like sequence immediately 5' to homologous or heterologous promoters conferred LPS-induced transcription, indicating that this NFIL-6-like consensus site was a transcriptional activator. Anti-C/EBP beta (NFIL-6) and anti-C/EBP delta (NFIL-6 beta) Abs identified both of these proteins in complexes formed between the NFIL-6-like element and mononuclear cell nuclear extracts. C/EBP delta (NFIL-6 beta) was not detected in complexes utilizing extracts from the IL-1 nonproducing T cell line. These data are consistent with the requirement for C/EBP beta (NFIL-6) and C/EBP delta (NFIL-6 beta) in the activation of murine IL-1 beta gene expression by endotoxin.

An NFIL-6 sequence near the transcriptional initiation site is necessary for the lipopolysaccharide induction of murine interleukin-1 beta.

We have analyzed the interleukin-1 beta (IL-1 beta) promoter region near the cap site. Specific DNA sequences required for lipopolysaccharide (LPS) induction within this region were identified using transfection of reporter plasmids that contained portions of the proximal IL-1 beta 5'-flanking sequence. An LPS-responsive activation area was localized between nucleotides -50 to -100, and down-regulating sequences were present between nucleotides -100 and -2,111. A NFIL-6 site between -92 and -84 was identified in the functionally active region. Base substitutions within this single NFIL-6 site in the context of a 4.1-kb IL-1 beta promoter segment resulted in dramatic reduction of LPS-induced gene transcription. Introduction of multimers of this NFIL-6 sequence immediately 5' to minimal homologous or heterologous promoters conferred LPS inducibility in each case. Anti-C/EBP beta (NFIL-6) and anti-C/EBP delta (NFIL-6 beta) antibodies identified both of these proteins in complexes formed between the NFIL-6 site and mononuclear cell nuclear extracts. These data show that the proximal NFIL-6 site is required for the activation of murine IL-1 beta gene expression by endotoxin.

Regulation of IL-1 gene expression: differential responsiveness of murine macrophage lines.

In order to begin to define the mechanisms by which lipopolysaccharide (LPS) regulates IL-1 gene expression, we have examined IL-1 RNA levels, the transcription rate of the IL-1 genes, and IL-1 mRNA stabilities in P388D1/C, RAW264.7, and murine peritoneal exudate cells (PEC). These experiments showed that total cellular IL-1 RNA levels and IL-1 transcription rates were dramatically upregulated in all three cell types. In all cases, IL-1 alpha and IL-1 beta cellular RNA levels and gene transcription rates were regulated in parallel. However, the profiles of IL-1 gene activation during the 24 h after LPS treatment differed in these three cell types. Additionally, culture in the presence of actinomycin D (Act D) showed differential stabilities of the IL-1 alpha and IL-1 beta RNAs in these cells. In peritoneal exudate cells, the half-lives (t1/2) of the IL-1 alpha and IL-1 beta RNAs were each > 8 h. In RAW264.7 cells, the stability of the IL-1 beta RNA was greater than the IL-1 alpha RNA (t1/2 > 8 h and approximately 6 h, respectively). In P388D1/C cells, the t1/2's of the IL-1 alpha and beta RNAs varied depending on the time of addition of actinomycin D. This and other data suggest that components of the IL-1 RNA catabolic pathway are labile and sensitive to treatment with actinomycin D. Together these data indicate that the two IL-1 genes show a diverse regulatory repertoire, even within related mononuclear phagocytic cells.

[Growth factors in human tooth development]. / Fattori di crescita nello sviluppo del dente umano.

Our research concerns the immunohistochemical localization of EGF and IGF-I receptors in the tooth germ, using monoclonal antibodies. The results show that in the early phases of human tooth development EGF and IGF-I receptors are present. At bud stage both receptors are localized at dental laminae level, in some epithelial cells of the tooth bud and in some mesenchymal cells. At cap stage the receptors are present in the outer and inner enamel epithelium, and in some cells of stellate reticulum. As far as concerns the mesenchymal cells, some cells of dental papilla in contact with enamel organ, are intensely positive. The immunopositivity is present also in some mesenchymal cells at follicular level. At late cap stage and at early bell stage receptors are not present at inner enamel epithelium level but they can be detectable in the mesenchyma of dental papilla and in some cells of the follicle. On the basis of these results it may be hypothesized that EGF and IGF-I can act as growth factors in the modulation of cellular proliferation and differentiation during the human tooth morphogenesis. Moreover, it is possible that these substances can play a role in the mesenchymal-epithelial interaction in the developing human tooth.