Regulation of IL-1-induced gingival collagenase gene expression by activator protein-1 (c-Fos/c-Jun).

Matrix metalloproteinase-1 is probably involved in the progression of periodontal disease. The aim of this study was to investigate whether IL-1beta stimulates the expression of the activator protein 1 (AP-1) transcription factor and, consequently, if the AP-1 transcription factor participates in the regulation of collagenase gene expression in human gingival fibroblast cells. In this study, we demonstrate that the concentration of the protein components of AP-1 transcription factor, c-Fos and c-Jun, is enhanced by IL-1beta both at mRNA and protein levels, utilizing Northern blot analysis, electrophoretic mobility gel shift assay and Western blot analysis. The IL-1beta stimulated the collagenase-CAT and AP-1-CAT activities in a dose dependent manner with respect to the amount of DNA used in transfections. Further, overexpression of c-Fos and c-Jun proteins revealed a dose-dependent transcriptional activation of the collagenase promoter. These findings, coupled with the existence of AP-1 consensus DNA binding sites on the collagenase gene promoter, show that regulation of collagenase gene expression by IL-1beta involves the transcription factor AP-1 in gingival fibroblasts.

Inhibition of gingival collagenase gene expression by dexamethasone.

Glucocorticoids are potent immunosuppressants shown to be effective in the treatment of inflammatory diseases. Reportedly, they work, in part, by inhibiting cytokines and other transcription factors including AP-1. In this study we investigated the mechanisms of efficient repression of collagenase gene expression by dexamethasone in the human gingival fibroblast. Northern analyses showed that IL-1-dependent collagenase mRNA production was significantly decreased in the presence of dexamethasone. The influence of dexamethasone on the transcription factor NF-kappaB, STAT3, and AP-1 was investigated by using the gel mobility shift assay with nuclear extracts prepared from the cells grown in the presence of dexamethasone. We observed that in addition to AP-1, binding of NF-kappaB and STAT3 to DNA was also decreased significantly. Additionally, dexamethasone induced the transcription of the I kappaB-alpha gene suggesting that in the presence of dexamethasone, NF-kappaB quickly reassociates with newly synthesized I kappaB-alpha and markedly reduces the amount of NF-kappaB. CAT transfection studies utilizing collagenase promoter demonstrated a dose-dependent transcriptional inhibition of IL-1-induced gingival collagenase gene expression by dexamethasone. These data reveal that collagenase gene expression can be regulated by the impairment of IL-1-stimulated NF-kappaB, STAT3, and AP-1 activities, and can highlight a possible molecular mechanism for the anti-inflammatory effects of glucocorticoids.

The effect of route of delivery on regression of abnormal cervical cytologic findings in the postpartum period.

OBJECTIVES: We sought to determine whether pregnant women with abnormal antepartum cervical cytologic findings differ in their postpartum rates of regression with respect to mode of delivery. STUDY DESIGN: Between 1990 and 1997, 446 pregnant women with antepartum abnormal cervical cytologic findings were identified. Complete demographic, clinical, and cytologic reports were available for 138 women. Papanicolaou smear data were collected and separated into three groups by use of the Bethesda classification system (atypical squamous cells of undetermined significance, low-grade squamous intraepithelial cells, and high-grade intraepithelial cells). Postpartum regression rates of antepartum Papanicolaou smears, with respect to degree of squamous epithelial cell abnormality and mode of delivery, were analyzed by Fisher's exact and Wilcoxon rank sum tests. RESULTS: Of the 138 women, 109 (79%) were delivered vaginally and 29 (21%) by cesarean section. No statistically significant difference was found between women delivered vaginally and those delivered by cesarean section with respect to age, parity, and smoking history within the three groups (atypical squamous cells of undetermined significance, low-grade squamous intraepithelial cells, and high-grade squamous intraepithelial cells). The overall postpartum regression rate for the 59 women with antepartum high-grade squamous intraepithelial cells was 48%. Of the 47 women with high-grade squamous intraepithelial cells who were delivered vaginally, 28 showed regression in the postpartum period versus none of the 12 women delivered by cesarean section (60% vs 0%, p < 0.0002). CONCLUSION: Postpartum spontaneous regression of Papanicolaou smears consistent with high-grade squamous intraepithelial cells occurs with increased frequency among women who are delivered vaginally versus by cesarean section.

Antizyme prevents ornithine decarboxylase-mediated cell death in human fibroblasts.

Ornithine decarboxylase (ODC) is a key enzyme of polyamine biosynthesis essential for growth-related cellular functions. Apart from its physiological role in cell proliferation, ODC also contributes to the induction of apoptosis under certain conditions, e.g. following growth factor withdrawal. The rate of cell death is a function of its enzyme activity, ODC activity is inhibited by a regulatory protein antizyme, also known to suppress polyamine uptake. We report that forced expression of antizyme prevents ODC-mediated cell death in human gingival fibroblasts under very low serum conditions. These data suggest an important antiapoptotic role for antizyme in cell survival.

Effect of Semicarpus anacardium on the cell cycle of DU-145 cells.

The nuts of Semicarpus anacardium (Anacardiaceae) are one of the most favoured medicine in the Indian System of Medicine for the management of arthritis and several other free radical mediated diseases. It is also recommended for the management of the breast cancer. In this report we have investigated its role on the cell cycle and cell viability on the DU-145 cells (transformed prostate cells) by flow cytometric technique. It was observed that the plant extract significantly arrests the cell cycle at G-1 stage, and induced apoptosis. On higher concentrations, it affects the cell viability. The response was dose dependent.

Nutrition and the geriatric implant patient.

The clinician must be cognizant of nutritional factors and deficiencies that can adversely affect implant surgery and subsequent prosthodontic rehabilitation for the geriatric patient. There are many nutrients, vitamins, and minerals that may be useful and even necessary for the geriatric implant patient. Dental implants and implant-retained and/or supported prostheses are viable treatment options for older patients when certain age-related and nutritional factors are considered.

Association of interleukin-1-induced, NF kappa B DNA-binding activity with collagenase gene expression in human gingival fibroblasts.

During earlier examination of interleukin-1 (IL-1)-induced matrix metalloproteinase gene expression in human gingival fibroblasts a highly induced immediate early gene, I kappa B-alpha, a NF kappa B DNA-binding inhibitor, was identified. The aim now was to investigate whether recombinant (r)IL-1 beta induces the stimulation of NF kappa B and its inhibitor proteins in human gingival fibroblasts and to understand if inhibition of its activity affects collagenase gene expression. Primary gingival fibroblasts (human) were treated with rIL-1 beta to determine the effect on NF kappa B-like DNA-binding activity. IL-1 induced the production of steady-state mRNA levels of I kappa B-alpha in the cultured fibroblasts. Nuclear run-on transcription studies demonstrated that rIL-1 induction of I kappa B-alpha may be transcriptionally regulated. Using electrophoretic mobility gel-shift assays it was shown that rIL-1 activates NF kappa B-like, DNA-binding activity in these fibroblasts. NF kappa B-like DNA-binding activity was rapidly induced and turned over in gingival fibroblasts with peak activity at 30 min after rIL-1 treatment. Further, treatment with chymotrypsin protease inhibitor and antioxidant inhibitor prevented IL-1-induced, NF kappa B-like, DNA-binding activity and collagenase mRNA production. When coupled with the existence of NF kappa B consensus DNA-binding sites on the collagenase gene promoter, these findings suggest that the stimulation of NF kappa B in gingival fibroblasts by rIL-1 could play an important part in the regulation of their collagenase gene expression. The ability of IL-1 to stimulate this expression may define a pivotal role for this cytokine in the pathogenesis of periodontitis.

Molecular cloning and sequencing of a human cDNA encoding ornithine decarboxylase antizyme.

We report the cloning of a cDNA encoding the human homolog of ornithine decarboxylase antizyme from a human gingival fibroblast cDNA library. The human antizyme is 84% identical to the rat sequence and shows almost no homology to the E. coli antizyme. Northern analysis studies show that this gene is expressed in both human gingival and synovial fibroblasts.

Mechanistic features associated with induction of metalloproteinases in human gingival fibroblasts by interleukin-1.

Human gingival fibroblasts were treated with recombinant interleukin-1 (IL-1) to determine the effect of this stimulus on the relative expression of collagenase (MMP-1), stromelysin (MMP-3) and plasminogen activator (PA) mRNA. The steady-state mRNA levels for these genes were determined on Northern blots. IL-1 induced steady-state levels of these mRNAs to different extents. Nuclear run-on transcription studies showed that IL-1 induction of neutral metalloproteinase may be transcriptionally regulated. Actinomycin D and protein kinase inhibitors decreased the mRNA production for all three metalloproteinases, whereas cycloheximide decreased the production of collagenase and stromelysin mRNA. Protein kinase inhibitors (H7/H8) decreased production of the three mRNAs to different extents. This study demonstrates a potentially important role for IL-1 in the regulation of metalloproteinase expression in human gingival fibroblasts. The ability of IL-1 to induce the expression of stromelysin, collagenase and PA may define a pivotal role for this cytokine in the pathogenesis of periodontitis.

Novel delayed-early and highly insulin-induced growth response genes. Identification of HRS, a potential regulator of alternative pre-mRNA splicing.

We have identified 41 novel and many previously known growth response genes induced in regenerating liver and insulin-treated Reuber H35 cells, a rat hepatoma cell line that grows in response to physiologic concentrations of insulin and retains some properties of regenerating liver. Although many genes are expressed similarly in the two systems, there are important differences in the kinetics of induction of some genes. These differences allowed us to identify and characterize novel genes that are highly insulin-induced and expressed as delayed-early genes in regenerating liver. Sequence analysis of CL-6, the most abundant insulin-induced gene, resulted in the identification of a highly hydrophobic hepatic protein. Sequence analysis of HRS, a highly insulin-induced delayed-early gene, demonstrated that it is a member of the family of regulators of alternative pre-mRNA splicing. Different forms of HRS mRNA are temporally regulated during the growth response, suggesting that HRS could autoregulate processing of its pre-mRNA. Given the dramatic increase in RNA production during late G1, proteins induced by mitogens like insulin that control RNA processing are likely to have important roles in cell cycle regulation.

Monoclonal antibodies to Escherichia coli ribosomal proteins L9 and L10. Effects on ribosome function and localization of L9 on the surface of the 50 S ribosomal subunit.

Monoclonal antibodies against Escherichia coli ribosomal proteins L9 and L10 were obtained and their specificity confirmed by Western blot analysis of total ribosomal protein. This was particularly important for the L9 antibody, since the immunizing antigen mixture contained predominantly L11. Each antibody recognized both 70 S ribosomes and 50 S subunits. Affinity-purified antibodies were tested for their effect on in vitro assays of ribosome function. Anti-L10 and anti-L9 inhibited poly(U)-directed polyphenylalanine synthesis almost completely. The antibodies had no effect on subunit association or dissociation and neither antibody inhibited peptidyltransferase activity. Both antibodies inhibited the binding of the ternary complex that consisted of aminoacyl-tRNA, guanylyl beta, gamma-methylenediphosphonate, and elongation factor Tu, and the binding of elongation factor G to the ribosome. The intact antibodies were more potent inhibitors than the Fab fragments. In contrast to the previously established location of L10 at the base of the L7/L12 stalk near the factor-binding site, the site of anti-L9 binding to 50 S subunits was shown by immune electron microscopy to be on the L1 lateral protuberance opposite the L7/L12 stalk as viewed in the quasisymmetric projection. The inhibition of factor binding by both antibodies, although consistent with established properties of L10 in the ribosome, suggests a long range effect on subunit structure that is triggered by the binding of anti-L9.

Probing the functional role and localization of Escherichia coli ribosomal protein L16 with a monoclonal antibody.

A monoclonal antibody specific for Escherichia coli ribosomal protein L16 was prepared to test its effects on ribosome function and to locate L16 by immunoelectron microscopy. The antibody recognized L16 in 50 S subunits, but not in 70 S ribosomes. It inhibited association of ribosomal subunits at 10 mM Mg2+, but not at 15 mM Mg2+. Poly(U)-directed polyphenylalanine synthesis and peptidyltransferase activities were completely inhibited when the L16 antibody was bound to 50 S subunits at a molar ratio of 1. There was no inhibitory effect on the binding of elongation factors or on the associated GTPase activities. Fab fragments of the antibody gave the same result as the intact antibody. Chemical modification of the single histidine (His13) by diethyl pyrocarbonate destroyed antibody binding. Electron microscopy of negatively stained antibody subunit complexes showed antibody binding beside the central protuberance of the 50 S particle on the side away from the L7/L12 stalk and on or near the interface between the two subunits. This site of antibody binding is fully consistent with its biochemical effects that indicate that protein L16 is essential for the peptidyltransferase activity activity of protein biosynthesis and is at or near the subunit interface.

Posttranscriptional mechanisms account for differences in steady state levels of insulin receptor messenger RNA in different cells.

Previous studies have indicated that, in general, the insulin receptor gene is expressed at a level in cells reflecting the level of insulin receptors on the cellular surface. For instance, insulin-responsive tissues, such as hepatocytes, express high levels of both insulin receptor protein and mRNA relative to less responsive cells, such as fibroblasts. Moreover, in the cells of a patient (Minn1) with severe insulin resistance and very low levels of insulin receptors, it has been shown that insulin receptor gene transcripts are virtually undetectable. Our earlier studies of the insulin receptor gene promoter suggested that the differences in the steady state level of insulin receptor gene transcripts in different cells could be transcriptionally mediated. In this study we have attempted to assess the relative contribution of transcriptional and posttranscriptional mechanisms in determining steady state levels of insulin receptor mRNA in various cells, including Minn 1 fibroblasts. Using nuclear run-on assays, we have determined that the level of nascent insulin receptor gene transcripts is roughly equal in different cells, including Minn1 fibroblasts. Therefore, transcriptional differences do not seem to account for the dramatic differences in steady state levels of insulin receptor mRNA in different cells, and there is no evidence in support of a transcriptional defect in Minn1's insulin receptor gene alleles. However, the rate of insulin receptor mRNA turnover varies significantly in different cells, ranging from an mRNA half-life of as little as 2 h in fibroblast and IM9 (lymphocytic) cells up to 8 h in HepG2 (liver) cells, and accounts in part for the observed differences in steady state insulin receptor mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

The gene encoding rat insulinlike growth factor-binding protein 1 is rapidly and highly induced in regenerating liver.

The liver is an epithelioid organ that can regenerate following partial hepatectomy. Although it is composed mainly of hepatocytes, it has a complex, multicellular architecture, implying that intercellular communications must exist during regeneration. As in other mitogen-stimulated cells, immediate-early growth response genes induced in the absence of prior protein synthesis are likely to play an important regulatory role in the regenerative process. Through differential screening of regenerating liver cDNA libraries, we found that one of the most highly expressed immediate-early genes in liver regeneration encodes the rat homolog of the low-molecular-weight insulinlike growth factor (IGF)-binding protein (IGFBP-1). This protein has been implicated in enhancing the mitogenic effect of IGF on tissues. IGFBP-1 gene induction is transcriptionally mediated and specific to regenerating liver, as the gene is not expressed in mitogen-stimulated fibroblasts. IGFBP-1 expression has been shown to increase under low-insulin conditions such as diabetes, and the complex regulation of expression is indicated by our finding that insulin treatment of H35 rat hepatoma cells, which induces proliferation, also causes a rapid decrease in transcription and expression of the IGFBP-1 gene. Of note, IGFBP-1 mRNA is abundant in fetal rat liver, implying that it participates in normal liver growth and development. Although regenerating liver cells continue to produce IGF-I, we did not detect IGF-I receptor mRNA during the first 24 h after hepatectomy. However, some IGFBPs may act to enhance the activity of IGF-I independently of IGF-I receptors. Thus, IGF-1 and IGFBPs may interact with hepatocytes or nonparenchymal liver cells, through either IGF-I or novel receptors. In this way, IGFBP-I and IGF-I could act in a paracrine and/or autocrine fashion in maintaining normal liver architecture during regeneration.

Characterization of the promoter region and 3' end of the human insulin receptor gene.

The insulin receptor is an essential protein present on the surface of virtually all cells. Little is known about the control of the level of this protein on cellular surfaces, but it has been found that the level of insulin receptor protein correlates roughly with the level of insulin receptor (IR) gene transcripts within cells. Although the protein-encoding region is only about 4000 base pairs (bps), there are multiple species of IR mRNA ranging in size from 5400 to 9400 bps. We have found that the variation in size of these transcripts is due to multiple 3' ends, presumably reflecting alternative polyadenylation, so that the final IR exon ranges in size from 1400 to 5400 bps. The IR gene promoter is like other housekeeping promoters in that it has no TATA or CAAT boxes, is extremely GC-rich, and has multiple transcriptional initiation sites primarily within a 300-bp GC-rich region. Reporter gene analysis using IR promoter-chloramphenicol acetyltransferase (HIRcat) fusion plasmids established regions responsible for promoter activity and verified the localization of the major IR gene transcriptional initiation sites. However, transfection with HIRcat plasmids containing regions from -153 to -1818 resulted in increased utilization of the most 5' IR gene mRNA initiation sites in transfected relative to untransfected cells. Reporter gene analysis also established that a region of the IR promoter and first exon containing all of the transcriptional initiation sites is more active in HepG2 than CV1 cells. Because the steady-state level of expression of the IR gene is much higher in HepG2 than CV1 cells, the results of the reporter gene analysis may reflect tissue-specific differences in IR gene transcription. Such tissue-specific transcriptional regulation would be a novel finding in a housekeeping promoter.

Insulin-receptor gene and its expression in patients with insulin resistance.

We studied the structure of the insulin-receptor gene in normal individuals and in four unrelated patients with leprechaunism (Minn-1, Ark-1, Ark-2, Can-1) and four unrelated patients with the type A syndrome of insulin resistance, both disorders associated with genetic alterations in affinity, binding capacity, and kinase activity of the insulin receptor. Genomic cloning and Southern blot analysis indicate that the normal human insulin-receptor gene is greater than or equal to 150 kilobases long and consists of a minimum of 17 exons, 6 in the genomic region of the alpha-subunit and 11 in the region of the beta-subunit. Three of the patients, one with leprechaunism and two with type A syndrome, have decreases in insulin-receptor mRNA but on genomic blot analysis have no obvious abnormalities in the insulin-receptor gene. No distinctive pattern of restriction-fragment-length polymorphisms or evidence for major insertion or deletion mutations of the insulin-receptor gene was found in any of the patients. These data indicate that the insulin-receptor gene is greater than 35 times larger than coding regions and has a complex structure. Although leprechaunism and type A syndrome are most likely due to defects in the structure and expression of the insulin-receptor gene, they are likely to be associated with specific point mutations rather than major changes in gene structure.

Probing ribosome function and the location of Escherichia coli ribosomal protein L5 with a monoclonal antibody.

A monoclonal antibody specific for Escherichia coli ribosomal protein L5 was isolated from a cell line obtained from Dr. David Schlessinger. Its unique specificity for L5 was confirmed by one- and two-dimensional electrophoresis and immunoblotting. The antibody recognized L5 both in 50 S subunits and 70 S ribosomes. Both antibody and Fab fragments had similar effects on the ribosome functions tested. Antibody bound to 50 S subunits inhibited their reassociation with 30 S subunits at 10 mM Mg2+ but not 15 mM, the concentration present for in vitro protein synthesis. The 70 S couples were not dissociated by the antibody. The antibody caused inhibition of polyphenylalanine synthesis at molar ratios to 50 S or 70 S particles of 4:1. The major inhibitory effect was on the peptidyltransferase reaction. There was no effect on either elongation factor binding or the associated GTPase activities. The site of antibody binding to 50 S was determined by electron microscopy. Antibody was seen to bind beside the central protuberance or head of the particle, on the side away from the L7/L12 stalk, and on or near the region at which the 50 S subunit interacts with the 30 S subunit. This site of antibody binding is fully consistent with its biochemical effects.

Monoclonal antibodies to epitopes in both C-terminal and N-terminal domains of Escherichia coli ribosomal protein L7/L12 inhibit elongation factor binding but not peptidyl transferase activity.

Two monoclonal antibodies against different epitopes in Escherichia coli ribosomal protein L7/L12, one within residues 74-120 and the other within residues 1-73, shown before to inhibit the binding of EF-G, have been tested for their effects on the binding to E. coli ribosomes of EF-Tu-aminoacyl-tRNA-GTP ternary complex and on peptidyl transferase activity. Both antibodies inhibit the binding of ternary complex and EF-Tu-dependent GTPase but have no inhibitory effect on peptidyl transferase activity. The inhibition of binding of both elongation factors is indicative of overlapping binding sites for EF-G and EF-Tu. The inhibition by both antibodies implies the contribution of both domains of L7/L12 to this binding site. This implies the location of one or more of the C-terminal domains of L7/L12 on the body of the 50S subunit. The absence of any inhibition of peptidyl transferase activity shows distinct separation of this site from the factor binding site.

Two monoclonal antibodies against Escherichia coli ribosomal protein L2 distinguish different locations for their respective epitopes in intact ribosomes.

Two monoclonal antibodies raised against intact Escherichia coli ribosomal protein L2 were isolated, affinity-purified, and characterized. One of the antibodies (Ab 5-186) recognizes an epitope within residues 5-186, and the other (Ab 187-272) recognizes an epitope within residues 182-272. Both antibodies strongly inhibit in vitro polyphenylalanine synthesis when they are first allowed to bind to 50 S subunits prior addition of 30 S subunits. However, only Ab 187-272 is inhibitory when added to preformed 70 S ribosomes. Ab 5-186 binds to 50 S subunits but not to 70 S ribosomes. Ab 187-272 does not cause dissociation of 70 S ribosomes under the ionic conditions of the assay for polyphenylalanine synthesis (15 mM magnesium), although at 10 mM magnesium it does cause dissociation. Both antibodies inhibit the reassociation of 50 S with 30 S subunits. Both antibodies strongly inhibit peptidyltransferase activity. The two antibodies differ in their effects on interactions with elongation factors Tu (EF-Tu) and G (EF-G). Neither antibody significantly inhibits EF-G-dependent GTPase activity, nor the binding of EF-G when the antibodies are incubated with 50 S subunits; however, Ab 187-272 causes a decrease in the binding of EF-Tu X aminoacyl-tRNA X GTP ternary complex and of EF-Tu-dependent GTPase when it is incubated with 70 S ribosomes. The Fab fragments of both antibodies had effects similar to the intact antibodies. The results show that monoclonal antibodies can be used to discriminate different regions of L2 and that EF-Tu and EF-G do not have identical ribosomal binding sites.