Interleukin 1 induces hypoxia-inducible factor 1 in human gingival and synovial fibroblasts.

Rheumatoid arthritis and periodontitis are inflammatory diseases modulated by proinflammatory cytokines [e.g. interleukin (IL-1) 1 and tumour necrosis factor alpha], which activate local fibroblasts to do the following: (1) proliferate, (2) induce gene expression and (3) produce destructive metalloproteinases. Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor (composed of HIF-1alpha and HIF-1beta/aryl hydrocarbon receptor nuclear transporter) that is modulated by hypoxia. HIF-1 binds to and induces several genes containing an HIF-1 consensus-binding site, including vascular endothelial growth factor and several glycolytic enzymes. Through differential screening of a human synovial fibroblast cDNA library, we identified HIF-1alpha as a clone up-regulated by IL-1. The mRNA for HIF-1alpha subunit was increased 3-4-fold by Northern blot analysis after cells had been incubated for 3 h in the presence of IL-1. In addition, IL-1 increased the binding of the heterodimer HIF-1 to the HIF consensus sequence. These results suggest that HIF-1 might have a role in inflammation, possibly in attempting to re-establish homoeostasis.

Induction of bone morphogenetic protein-2 by interleukin-1 in human fibroblasts.

Rheumatoid arthritis and periodontitis are chronic inflammatory diseases associated with tissue destruction that is mediated in part by elevated levels of cytokines (e.g., interleukin-1 and tumor necrosis factor). Differential screening of a human synovial fibroblast cDNA library for interleukin-1 induced genes revealed a clone identical to the gene encoding human bone morphogenetic protein-2. Northern blot analysis of human synovial fibroblast mRNA confirmed up-regulation of bone morphogenetic protein-2 in the presence of interleukin-1. Utilizing a specific antibody, levels of bone morphogenetic protein-2 protein in conditioned medium from synovial fibroblasts were also up-regulated in the presence of interleukin-1. This is the first report of the production of bone morphogenetic protein-2 by synovial fibroblasts, and the first report of its up-regulation in response to interleukin-1. However, interleukin-1 did not induce bone morphogenetic protein-2 mRNA in human gingival fibroblasts.

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.

Expression of mRNA for serglycin core protein and other platelet alpha granule proteins is increased in human erythroleukemia cells by phorbol myristate acetate.

This study has determined the effects of phorbol-12-myristate-13-acetate (PMA) and dimethylsulfoxide (DMSO) on mRNA levels for the serglycin proteoglycan core protein in human erythroleukemia (HEL) cells. We have compared these changes to those for mRNA for other proteins which are known to be synthesized by HEL cells and megakaryocytes and are known to be localized to alpha granules within platelets. PMA caused a large increase in mRNA for serglycin within two hours of treatment of the cells, and the increase persisted for at least 72 hours. DMSO did not cause a significant change in mRNA levels. mRNA for platelet factor 4, transforming growth factor-beta, and P-Selectin (PADGEM, GMP-140) were also increased by PMA treatment. The mRNA for platelet factor 4 was substantially reduced in the presence of DMSO. The increase of mRNA for serglycin induced by PMA was consistent with our previous observation that synthesis of proteoglycans from [35S]sulfate was greatly stimulated by PMA in HEL cells. The data suggest that up-regulation of synthesis of proteoglycans is induced by PMA in cells which have the capacity to differentiate along the megakaryocytic lineage, as opposed to cell lines such as HL-60 in which proteoglycan synthesis is reduced in the presence of this differentiation-inducing agent.

P-selectin mRNA is expressed at a later phase of megakaryocyte maturation than mRNAs for von Willebrand factor and glycoprotein Ib-alpha.

The assembly of alpha-granules occurs exclusively in megakaryocytes because platelets have limited capacity for the synthesis of macromolecules. Thus far, alpha-granule development in megakaryocytes has been primarily evaluated by ultrastructural studies. The aim of the study was to obtain molecular and biochemical evidence for the expression of selected alpha-granule proteins in megakaryocytes. Guinea pig megakaryocytes were purified and separated into subgroups at different phases of maturation by the Celsep procedure (Schick et al. Blood 1989;73:1801-8). Guinea pig-specific probes for P-selectin, von Willebrand factor (vWF), glycoprotein Ib-alpha (GpIb-alpha), and phosphoglycerate kinase were prepared by using the polymerase chain reaction. By Northern blot analysis, P-selectin messenger ribonucleic acid (mRNA) was primarily expressed in the mature megakaryocyte Celsep subgroup, whereas vWF and GpIb-alpha mRNA were expressed at all phases of megakaryocyte maturation. In situ hybridization confirmed that P-selectin mRNA was primarily expressed at later stages of cytoplasmic maturation: 14% +/- 6.2% of stage I, 35.5% +/- 6.1% of stage II, 72% +/- 5.2% of stage III, and 47.0% +/- 3.3% of stage IV megakaryocytes expressed P-selectin mRNA. Thus, the expression of mRNA for P-selectin appeared to peak in stage III cells. In contrast vWF mRNA was expressed in immature megakaryocytes and persisted throughout megakaryocyte maturation. In situ hybridization did not demonstrate a relationship between the expression of mRNA for P-selectin or vWF with megakaryocyte ploidy.(ABSTRACT TRUNCATED AT 250 WORDS)

Otoacoustic emissions and auditory brainstem responses in the newborn.

The auditory function of 370 infants, drawn from both low and high risk groups, was tested before postnatal discharge using three tests: standard auditory brain stem responses (ABR), automated analysis of ABR, and automated analysis of evoked otoacoustic emissions (OAE). All infants failing any neonatal test had further audiological evaluation. Follow up information was also available on those who passed neonatal tests. Automated OAE testing of both ears was quickest (median 12.5 minutes) and least invasive (no scalp electrodes). Bilateral failure rates (and upper 95% confidence limits) with a stimulus 35-36 dB above normal hearing threshold level (nHL) were 3.0% (4.6) with automated OAE, 3.2% (5.1) with ABR, and 2.7% (4.4) with automated ABR. Automated OAE was the test most sensitive for subsequently confirmed hearing impairment. Sequential testing with automated OAE followed, in those failing this test, by automated ABR would have provided a screening test for substantial hearing impairment with a specificity greater than 99% in this population. Possible application as a universal screen is discussed.

Structure, spatial, and temporal expression of two sea urchin metallothionein genes, SpMTB1 and SpMTA.

The metallothionein-B genes of the sea urchin Strongylocentrotus purpuratus encode a metallothionein (MT) isoform distinguishable from the MTA isoform. The MTB subfamily consists of at least two genes, MTB1 and MTB2, and possibly two to three others. The unique MTB1 and MTA genes have a high degree of identity but diverge in structural detail and expression. Transcripts of the MTA, MTB1, troponin C Spec 1, and CyIIIa actin genes begin simultaneously to accumulate at an early blastula stage. MTB1 mRNA becomes localized in the embryonic gut and oral ectoderm, whereas MTA, Spec 1, and CyIIIa actin mRNAs are spatially restricted to the aboral ectoderm. Several DNA elements are localized at the same positions in the MTB1 and MTA genes: these include respective CATA and TATA boxes, two metal response elements, and three distinct upstream DNA elements that are also present, and in the same order, in the Spec 1 gene promoter. A heptameric sequence, element A, is present at two sites each in the Spec 1 and CyIIIa actin genes, five sites in MTA, but only one site in MTB1. Most strikingly, the first intron of MTA contains elements not found in the MTB1 introns, including a consensus metal response element, an element A, and the P3A site demonstrated in the CyIIIa actin gene to be linked to the regulation of spatial expression.

Structure of an ectodermally expressed sea urchin metallothionein gene and characterization of its metal-responsive region.

The metallothionein-A gene in the metallothionein gene family of the sea urchin Strongylocentrotus purpuratus (SpMTA gene) was sequenced and found to contain three coding exons plus a 3' entirely noncoding exon. Putative alpha and beta MT domains were encoded, by its exons 2 and 3, respectively, in reverse of the order in vertebrate metallothionein genes. The SpMTA promoter was characterized through the expression of recombinant constructs containing various portions of the proximal 678-base-pair (bp) 5'-flanking region of the SpMTA gene. Zygotes injected with constructs were cultured to the blastula stage in the presence of a heavy-metal chelator and then incubated in the presence or absence of cadmium. The longest constructs were expressed only when heavy-metal ion was present. Two putative metal-responsive elements (MREs a and b) within 240 bp of the transcription start site resembled mammalian MREs in their critical 8-bp cores (TGCRCNCS) and in their locations relative to each other and to the TATA box. Elimination of activity by site-specific mutations in MREs a and b, separately or in both, identified them as metal regulatory elements. Thus, MRE recognition in this invertebrate resembles that in vertebrates. Upstream sites with single-mismatched MREs neither acted as MREs nor amplified the activity of MREs a and b. The SpMTA, Spec1, and CyIIIa actin genes, which have the same ectodermal specificity, have common DNA elements at relatively similar locations in their promoter regions; however, these elements are insufficient in themselves to promote gene expression.

Interaction of bovine factor XIIa with an inhibitor from bovine plasma.

An inhibitor of factor XIIa has been purified from bovine plasma and characterized (Thornton, R.D. and Kirby, E.P. (1987) J. Biol. Chem. 262, 12714-12721). This inhibitor interacts with XIIa to form a very stable complex with a 1:1 stoichiometry. The active site of XIIa, located on the light chain, is directly involved in the interaction, and complex formation between factor XIIa inhibitor and XIIa can be blocked by diisopropyl fluorophosphate, corn trypsin inhibitor, or the chromogenic substrate S2302. Incubation of the complex with excess XIIa does not result in cleavage of the complex. The complex does not spontaneously dissociate and is stable to boiling, SDS, thiocyanate, acid, and hydroxylamine or Tris at pH 7-10. In addition to complex formation, a cleaved form of factor XIIa inhibitor can be observed. We suggest that the inhibitor is acting as a mechanism-based inactivator, using the criteria of time-dependent inactivation under pseudo-first-order conditions, 1:1 stoichiometry, active site involvement, kinetic protection by substrate or by an active site inhibitor, and partitioning between cleavage of factor XIIa inhibitor and inactivation by complex formation.

Isolation and characterization of an inhibitor of factor XIIa from bovine plasma.

An inhibitor of factor XIIa has been purified to homogeneity from bovine plasma. The purification steps included precipitation of contaminating proteins with polyethylene glycol and chromatography on DEAE-cellulose, Affi-Gel blue, and immobilized wheat germ lectin. The apparent molecular weight of the XIIa inhibitor (called INH1) was 85,000, reduced, and 92,000, nonreduced, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The extinction coefficient (E0.1%(280)) of INH1 is 1.3, and the protein contains 17.7% carbohydrate. Purified antibody to INH1 raised in either rabbits or chickens formed a precipitin line of identity with purified INH1 and a component of bovine plasma, but there was no reaction with purified human inhibitors or with any component of human plasma. INH1 inhibits bovine and human XIIa, bovine and human C1-esterase, and human kallikrein, but does not inhibit bovine kallikrein, bovine trypsin, human plasmin, or human thrombin. This activity is similar to that of C1-inhibitor but different from antithrombin III, alpha 2-antiplasmin, or alpha 1-protease inhibitor. INH1 at a physiological concentration (0.47 microM) causes rapid inactivation of XIIa. The two molecules react in a 1:1 stoichiometry with a second-order rate constant of 1.23 X 10(6) M-1 min-1.

Human parotid alpha-amylase secretion as a function of chronic hyperbaric exposure.

Secretion of alpha-amylase by the human parotid gland increased significantly during eight days of hyperbaric exposure. This hyperactivity of the parotid gland presumably resulted from increased autonomic nervous system (ANS) activity attributable to (1)psychological stress in the form of anticipation; (2) dive-related factors, i.e., hyperoxia, PN2, physical stress; or (3) a combination of both. The etiology of the effect must await additional studies, but a consistent and significant elevation in alpha-amylas secretion was found. This previously undescribed effect of hyperbaric exposure indicates that parotid alpha-amylase sampling holds promise as a noninvasive means of monitoring physical and psychological stress, and as an indirect measure of ANS tone.

Comparison of a direct latex-agglutination technic with the tanned red cell hemagglutination inhibition immunoassay (TRCHII) for semiquantitation of fibrinogen/fibrin degradation products.

A new latex-agglutination kit for rapid screening for fibrinogen/fibrin degradation products (FDP/fdp) has been compared with a standard tanned red cell hemagglutination inhibition immunoassay (TRCHII). The latex-agglutination test results agreed with the TRCHII results for 489 of 588 samples (83%). FDP/fdp values were elevated by the latex-agglutination kit when normal by the TRCHII for 79 (13%) samples, and the reverse was true for 20 (3%) samples. Serial dilution of serum sample allowed reliable semiquantitation of FDP/fdp levels compared with TRCHII results (r=less than .001). The latex-agglutination test gave FDP/fdp values of larger than or equal to 10 mug. per ml. for sera from 11 of 13 patients who had acute pulmonary embolism and for only one of 24 normal control samples. The direct latex-agglutination kit for FDP/fdp appears to have appropriate sensitivity to serve as a screening test for acute pulmonary embolism in patients not receiving heparin therapy.