Production of matrix metalloproteinase-9 in lipopolysaccharide-stimulated human amnion occurs through an autocrine and paracrine proinflammatory cytokine-dependent system

The objective of this study was to determine the presence of autocrine/paracrine regulation of matrix metalloproteinase-9 (MMP-9) expression mediated by proinflammatory cytokines in human fetal membranes. Fetal membranes obtained from women who underwent cesarean delivery before labor were manually separated into amnion and chorion layers and maintained in culture. These explants were stimulated with tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), and either lipopolysaccharide (LPS) alone or LPS with anti-TNFalpha or anti-IL-1beta-neutralizing antibodies. Levels of proMMP-9 in culture media were evaluated by zymography. Enzyme-linked immunosorbant assay was performed to measure the quantity of IL-1beta, TNFalpha, and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) after LPS stimulation. ProMMP-9 activity was upregulated after stimulation of the amnion by LPS, TNFalpha, and IL-1beta. The increased activity of proMMP-9 resulting from LPS stimulation in the amnion was blocked by the addition of TNFalpha neutralizing antibody but not with anti-IL-1beta. No significant effect of LPS, TNFalpha, or IL-1beta on proMMP-9 expression was observed in the chorion; however, the chorion produced both cytokines when stimulated with LPS. In contrast, TIMP-1 levels remained unchanged in all cultures incubated in the presence of LPS. Therefore, these data indicate that proMMP-9 is produced by the amnion but not the chorion in response to LPS. Because anti-TNFalpha-neutralizing antibody inhibits proMMP-9 activity in the amnion, TNFalpha appears to upregulate proMMP-9 production by the amnion in an autocrine fashion. Meanwhile, TNFalpha and IL-1beta produced by the chorion may upregulate amnionic proMMP-9 production in a paracrine manner.     

Modeling alpha-helical coiled-coil interactions: the axial and azimuthal alignment of 1B segments from vimentin intermediate filaments

Attempts at predicting the relative axial alignments of fibrous protein molecules in filamentous structures have relied upon representing the (multichain) molecular structure by a one-dimensional sequence of amino acids. Potential intermolecular ionic and apolar interactions were counted and determined as a function of the relative axial stagger between the molecules. No attempts were made to consider the azimuthal aspect of the interacting molecules and neither were apolar or ionic energy terms used. Surprisingly, this simple approach proved remarkably informative and yielded accurate predictions of the axial periods present. However, a more comprehensive analysis involving the energetics of aggregation taking due regard for the relative azimuths of the molecules as well as their separation should decrease the noise level in the calculations and reveal other pertinent information. Toward that end, we have modeled the interaction between two alpha-helical coiled-coil segments in intermediate filament molecules (1B segments from human vimentin). The relative axial alignment and polarity of the molecules is already known from detailed crosslinking studies and this provides a criterion against which the success (or otherwise) of the modeling can be judged. The results confirm that an antiparallel alignment of two 1B segments is preferred over any of the parallel options (as observed experimentally). The calculated axial alignment, however, is not identical to that observed from detailed crosslinking studies indicating that other parts of the molecule (probably the head and tail domains as well as other coiled-coil segments) have a crucial role in determining the precise mode of axial aggregation. The results also show that the apolar interactions seem to be significantly less important in the alignment process than the ionic ones. This is consistent with the observation of a well-defined period in the linear disposition of the charged (but not apolar) residues along the length of the outer surface of the vimentin molecule.     

Macrofibril assembly in trichocyte (hard alpha-) keratins

Early electron microscope studies of developing wool and hair established that trichocyte (hard alpha-) keratin fibers have a composite structure in which filaments, subsequently shown to belong to the class of intermediate filaments (IF), were embedded in a matrix of sulfur-rich proteins. These studies also showed that the IF aggregate in a variety of ways to form what have been termed macrofibrils. Assembly into sheets appears to be an important initial factor in aggregation, and in the present contribution the structural principles governing sheet formation are formulated and specific models for the interaction between neighboring IF in a sheet are proposed, based on existing X-ray diffraction, electron microscope, and crosslinking data. All of the trichocyte keratins so far examined by electron microscopy exhibit similar filament/matrix textures and the mechanism of sheet formation proposed here is likely to have general applicability.     

Feasibility of using GFP-expressing Escherichia coli, coupled with fluorimetry, to determine protozoan ingestion rates

The feasibility of using a live Escherichia coli population, which had been engineered to express the green fluorescent protein (GFP), coupled with fluorimetry, was tested as a means for determining protozoan ingestion rates. Its potential use was based on evidence that once cells are acidified, e.g. in a food vacuole, the fluorescence is lost. Of the 29 protozoa tested, over 85% ingested the GFP-expressing E. coli and a detailed experiment with the ciliate Tetrahymena pyriformis was carried out, principally to assess the performance of the live bacterium against two commonly used surrogate prey, i.e. fluorescently labelled bacteria (FLB) and fluorescently labelled microspheres (FLMs). A decrease in GFP-expressing E. coli fluorescence and, hence, concentration, was recorded by fluorimetry and epifluorescence microscopy, with calculated ingestion rates being equivalent. A higher ingestion rate was determined by counting the number of fluorescent E. coli within the ciliate over 120 s, but this was equivalent to that obtained for the stained E. coli using the same direct method of analysis. However, the ciliate was shown to process the stained and unstained E. coli cells differently, with only the latter resulting in an increase in ciliate abundance.     

A second-generation genomewide screen for asthma-susceptibility alleles in a founder population

A genomewide screen for asthma- and atopy-susceptibility loci was conducted, using 563 markers, in 693 Hutterites who are members of a single 15-generation pedigree, nearly doubling the sample size from the authors' earlier studies. The resulting increase in power led to the identification of 23 loci in 18 chromosomal regions showing evidence for linkage that is, in general, 10-fold more significant (P<.001 vs. P<.01) than the linkages reported previously in this population. Moreover, linkages to loci in 11 chromosomal regions were identified for the first time in the Hutterites in this report, including five regions (5p, 5q, 8p, 14q, and 16q) showing evidence both of linkage, by the likelihood ratio (LR) chi(2), and of disequilibrium, by the transmission/disequilibrium test. A region on chromosome 19 continues to show evidence for linkage, by both tests, in this study. Studies of 17 candidate genes provide evidence for association with variation in the IL4RA gene (16p12), the HLA class II genes (6p21), and the interferon-alpha gene cluster (9p22), but the lack of evidence for linkage in these regions by the LR chi(2) test suggests that these are minor susceptibility loci. A polymorphism in the CD14 gene is in linkage disequilibrium with an as yet unidentified susceptibility allele in the 5q cytokine cluster, a region showing evidence for linkage among the Hutterites. Finally, 10 of the regions showing evidence for linkage in the Hutterites have shown evidence of linkage to related phenotypes in other genome screens, suggesting that these regions may contain common alleles that have relatively large effects on asthma and atopy phenotypes in diverse populations.     

Ribozymes as tools for therapeutic target validation in arthritis

In this paper we describe a method for validating therapeutic gene targets in arthritic disease. Ribozymes are catalytic oligonucleotides capable of highly sequence-specific cleavage of RNA. We designed ribozymes that cleave the mRNA encoding stromelysin, a matrix metalloproteinase implicated in cartilage catabolism. Ribozymes were initially screened in cultured fibroblasts to identify sites in the mRNA that were accessible for binding and cleavage. Accessible sites for ribozyme binding were found in various regions of the mRNA, including the 5' untranslated region, the coding region, and the 3' untranslated region. Several ribozymes that mediated sequence-specific and dose-dependent inhibition of stromelysin expression were characterized. Site selection in cell culture was predictive of in vivo bioactivity. An assay for measuring cartilage catabolism in rabbit articular cartilage explants was developed. Ribozymes inhibited IL-1-stimulated stromelysin mRNA expression in articular cartilage explants, yet failed to inhibit proteoglycan degradation. This indicated that up-regulation of stromelysin was not essential for IL-1-induced cartilage catabolism. Broad applications of this approach in therapeutic target validation are discussed.     

In vitro assembly and structure of trichocyte keratin intermediate filaments: a novel role for stabilization by disulfide bonding

Intermediate filaments (IF) have been recognized as ubiquitous components of the cytoskeletons of eukaryotic cells for 25 yr. Historically, the first IF proteins to be characterized were those from wool in the 1960s, when they were defined as low sulfur keratins derived from "microfibrils." These proteins are now known as the type Ia/type IIa trichocyte keratins that constitute keratin IF of several hardened epithelial cell types. However, to date, of the entire class of >40 IF proteins, the trichocyte keratins remain the only ones for which efficient in vitro assembly remains unavailable. In this paper, we describe the assembly of expressed mouse type Ia and type IIa trichocyte keratins into IF in high yield. In cross-linking experiments, we document that the alignments of molecules within reduced trichocyte IF are the same as in type Ib/IIb cytokeratins. However, when oxidized in vitro, several intermolecular disulfide bonds form and the molecular alignments rearrange into the pattern shown earlier by x-ray diffraction analyses of intact wool. We suggest the realignments occur because the disulfide bonds confer substantially increased stability to trichocyte keratin IF. Our data suggest a novel role for disulfide bond cross linking in stabilization of these IF and the tissues containing them.     

Coiled-coil trigger motifs in the 1B and 2B rod domain segments are required for the stability of keratin intermediate filaments

Many alpha-helical proteins that form two-chain coiled coils possess a 13-residue trigger motif that seems to be required for the stability of the coiled coil. However, as currently defined, the motif is absent from intermediate filament (IF) protein chains, which nevertheless form segmented two-chain coiled coils. In the present work, we have searched for and identified two regions in IF chains that are essential for the stability necessary for the formation of coiled-coil molecules and thus may function as trigger motifs. We made a series of point substitutions with the keratin 5/keratin 14 IF system. Combinations of the wild-type and mutant chains were assembled in vitro and in vivo, and the stabilities of two-chain (one-molecule) and two-molecule assemblies were examined with use of a urea disassembly assay. Our new data document that there is a region located between residues 100 and 113 of the 2B rod domain segment that is absolutely required for molecular stability and IF assembly. This potential trigger motif differs slightly from the consensus in having an Asp residue at position 4 (instead of a Glu) and a Thr residue at position 9 (instead of a charged residue), but there is an absolute requirement for a Glu residue at position 6. Because these 13 residues are highly conserved, it seems possible that this motif functions in all IF chains. Likewise, by testing keratin IF with substitutions in both chains, we identified a second potential trigger motif between residues 79 and 91 of the 1B rod domain segment, which may also be conserved in all IF chains. However, we were unable to find a trigger motif in the 1A rod domain segment. In addition, many other point substitutions had little detectable effect on IF assembly, except for the conserved Lys-23 residue of the 2B rod domain segment. Cross-linking and modeling studies revealed that Lys-23 may lie very close to Glu-106 when two molecules are aligned in the A(22) mode. Thus, the Glu-106 residue may have a dual role in IF structure: it may participate in trigger formation to afford special stability to the two-chain coiled-coil molecule, and it may participate in stabilization of the two-molecule hierarchical stage of IF structure.     

Acute toxicology and pharmacokinetic assessment of a ribozyme (ANGIOZYME) targeting vascular endothelial growth factor receptor mRNA in the cynomolgus monkey

The potential acute toxicity of a ribozyme (ANGIOZYME) targeting the flt-1 vascular endothelial growth factor (VEGF) receptor mRNA was evaluated in cynomolgus monkeys following i.v. infusion or s.c. injection. ANGIOZYME was administered as a 4-hour i.v. infusion at doses of 10, 30, or 100 mg/kg or a s.c. bolus at 100 mg/kg. End points included blood pressure, electrocardiogram (ECG), clinical chemistry, hematology, complement factors, coagulation parameters, and ribozyme plasma concentrations. ANGIOZYME was well tolerated, with no drug-associated morbidity or mortality. There was no clear evidence of ANGIOZYME-related adverse effects in this study. Slight increases in spleen weight and lymphoid hyperplasia were observed in several animals. However, these changes were not dose dependent. Steady-state concentrations of ANGIOZYME were achieved during the 4-hour infusion of 10, 30, or 100 mg/kg. Dose-dependent elimination of ANGIOZYME was observed, with faster clearance at the two highest doses. ANGIOZYME was slowly absorbed after s.c. administration, resulting in steady-state concentrations for the 9-hour sampling period. Monkeys in this toxicology study received significant plasma ANGIOZYME exposure by both the s.c. and i.v. routes.     

Reflections on the implications of thresholds of mutagenic activity for the labelling of chemicals by the European Union

During the course of the safety evaluation and regulatory control of chemicals it is important to distinguish between "potential hazard" and "actual risk" of exposure to toxins. In the case of DNA reactive chemicals, it has been prudent to assume that hazard is expressed as risk at low exposure concentrations. However, analysis of the dose-response relationships of both DNA reactive and non-DNA reactive genotoxins (e.g., aneugens) indicate that there are exposure concentrations below which protective mechanisms such as DNA repair activity and the presence of multiple targets may lead to the prediction of no risk until threshold concentrations are achieved. Current European Union management procedures for mutagenic chemicals are based predominantly upon hazard assessment rather than assessment of actual risk under likely exposure scenarios. As our knowledge of protective mechanisms increases, the time is now appropriate to undertake a re-evaluation of European Union criteria and to base the clarification mutagenic chemical more firmly upon the basis of actual risks to the human population and to the environment.