Preterm premature rupture of membranes without labor is not associated with increased levels of matrix metalloproteinase-9 protein.

OBJECTIVES: Preterm premature rupture of membranes (PROM) accounts for 30-40% of all preterm births. The objectives of this study were to determine whether matrix metalloproteinase-9 (MMP-9) is increased in preterm PROM fetal membranes, whether labor or gestational age affects expression, and whether the increase is localized to the rupture site or is membrane-wide. METHODS: Fetal membranes were collected from 15 pregnancies complicated by preterm PROM and 26 control cases, which delivered at term or preterm without PROM. The preterm PROM cases represented both patients who labored and those who did not. Membrane samples at the rupture site and a remote site (approximately > 5 cm) were analyzed for MMP-9 protein and enzymatic activity by Western blot and gelatin zymography, respectively. RESULTS: MMP-9 levels in fetal membranes were similar at both the rupture and the remote sites. The highest levels of total MMP-9 protein were found in preterm PROM patients with labor (p < 0.05) and were increased four-fold over protein levels in non-laboring preterm PROM patients delivered by Cesarean section (p < 0.001). In preterm PROM patients without labor, levels of MMP-9 protein were similar to those of non-laboring patients at term and preterm. Zymography correlated with protein results in all membranes. CONCLUSIONS: Preterm PROM without labor is not associated with increased membrane levels of MMP-9 protein, suggesting that its local elevation does not play a role in early membrane rupture.

Analysis of genomic instability using multiple assays in a patient with Rothmund-Thomson syndrome.

We report on a patient with Rothmund-Thomson syndrome (RTS) whose cytogenetic evaluation showed a normal karyotype with no evidence of trisomy mosaicism or chromosomal rearrangements. Cultured lymphocytes from the patient, her mother, and a control exposed to mitomycin C and diepoxybutane did not show increased sensitivity to the dialkylating agents. Unlike some previous reports, we found no evidence of a deficiency in nucleotide excision repair, as measured with the functional unscheduled DNA synthesis assay. Glycophorin A analysis of red blood cells for somatic mutation revealed suspiciously high frequencies of both allele loss and loss-and-duplication variants in the blood of the patient, a pattern consistent with observations in other RecQ-related human diseases, and evidence for clonal expansion of a mutant clone in the mother. Discrepant results in the literature may reflect true heterogeneity in the disease or the fact that a consistent set of tests has not been applied to RTS patients.

Elevated DNA excision repair capacity in the extraembryonic mesoderm of the midgestation mouse embryo.

In order to determine whether there is differential cell-type-specific DNA repair we measured the nucleotide excision repair capacity of the four distinct cell lineages that comprise the extraembryonic yolk sac using the unscheduled DNA synthesis assay. Yolk sacs from mouse embryos at 11.5-12.5 days gestation were microdissected to yield purified trophoblast, parietal endoderm, mesoderm, and visceral endoderm, as well as fetal skin fibroblasts which were then grown as primary explants. At this midgestational stage of development, the yolk sac provides essential functions for the sustenance of the embryo while the complex process of organogenesis is proceeding in the liver, kidney, and gut. Trophoblast giant cells, parietal endoderm, and visceral endoderm all demonstrated low levels of unscheduled DNA synthesis consistent with levels measured in adult mouse skin fibroblasts. As has previously been documented, embryonic mouse skin fibroblasts were reproducibly 2- to 3-fold higher than adult mouse skin fibroblasts in levels of DNA excision repair. The extraembryonic mesoderm, however, displayed a statistically significant level of unscheduled DNA synthesis 10-fold higher than adult mouse skin fibroblasts or the other lineages of the midgestation yolk sac. Further, the S-indexes of these lineages were also determined to assess the possible relevance of differential repair to the proliferative status of the cells. These data demonstrate that DNA excision repair capacity is lineage-specific during embryogenesis in the mouse. These studies may begin to provide a context for understanding the perplexing developmental aspects such as the characteristic congenital abnormalities associated with the human heritable DNA repair deficiency diseases.

Novel flow-cytometric method for separating cell types in differentiated F9 embryoid bodies.

The differentiation of F9 teratocarcinoma cells mimics the formation of a mouse embryonic tissue, the primitive endoderm. In vitro, small aggregates of F9 cells, termed embryoid bodies, differentiate in response to retinoic acid and develop a surface epithelium that is characterized by the production of alpha-fetoprotein. In the present study, cellular autofluorescence profiles obtained by fluorescence-activated embryoid bodies were composed of a single type of cell. In contrast, retinoic acid-induced embryoid bodies were composed of two cell types: a major population displaying autofluorescence levels similar to those of cells from undifferentiated embryoid bodies and a second population displaying higher autofluorescence. RNA analyses demonstrated that the transcription of alpha-fetoprotein was associated only with the more highly autofluorescent population, indicating that flow cytometry provides a novel mechanism for the separation of undifferentiated cells from differentiated endoderm cells in F9 embryoid bodies.

Evolution of murine alpha 1-proteinase inhibitors: gene amplification and reactive center divergence.

The organization and sequence of genes encoding the alpha 1-proteinase inhibitor (alpha 1PI), a major serine proteinase inhibitor of the mammalian bloodstream, have been compared in several species, including murine rodents (genus Mus). Analysis of gene copy number indicates that amplification of alpha 1PI genes occurred at some time during evolution of the Mus genus, leading to fixation of a family of about three to five genes in several existing species (e.g., M. domesticus and M. saxicola), and only a single gene in others (e.g., M. caroli). A phylogeny for the various mammalian alpha 1PI mRNAs was constructed based upon synonymous substitutions within coding regions. The mRNAs in different murine species diverged from a common ancestor before the formation of the first species lineages of the Mus genus, i.e., about 10-13 million years ago. Thus, alpha 1PI gene amplification must have occurred prior to Mus speciation; gene families were retained in some, but not all, murine species. The reactive center region of the alpha 1PI polypeptide, which determines target protease specificity, has diverged rapidly during evolution of the Mus species, but not during evolution of other mammalian species included in the analysis. It is likely that this accelerated evolution of the reactive center, which has been noted previously for serine proteinase inhibitors, was driven by some sort of a positive Darwinian selection that was exerted in a taxon-specific manner. We suggest that evolution of alpha 1PI genes of murine rodents has been characterized by both modification of gene copy number and rapid reactive center divergence. These processes may have resulted in a broadened repertoire of proteinase inhibitors that was evolutionarily advantageous during Mus speciation.

Highly conserved upstream regions of the alpha 1-antitrypsin gene in two mouse species govern liver-specific expression by different mechanisms.

alpha 1-Antitrypsin (AT), the major elastase inhibitor in mammalian serum, is produced primarily in the liver. We have characterized AT gene structure and expression in the mouse species Mus caroli, which expresses high levels of AT in the kidneys as well as in the liver. Analysis of cDNA and genomic clones showed that the AT gene in M. caroli exhibits high sequence homology (greater than 90%) to the gene in laboratory mice (M. domesticus) throughout the coding and 5'-flanking regions. Despite this extensive sequence conservation, the functional organization of cis-acting regulatory elements governing liver-specific expression is strikingly different between these species. Transient-transfection assays showed that the proximal region of the M. caroli promoter (i.e., between -120 and -2 relative to the transcriptional start site) is 10-fold more active than the analogous region of M. domesticus in driving the expression of an indicator gene in cultured liver cells. The increased activity of the proximal region of the M. caroli AT promoter appears to be the result of one or both of the two base substitutions at positions -46 and -48. The weak proximal promoter in M. domesticus is compensated for by the presence of upstream, liver-specific enhancers between -199 and -520; the analogous region in M. caroli is inactive. Thus, during the course of evolution, the modest 7% sequence divergence that has occurred between the 5'-flanking regions of the AT genes in these two species has generated distinct, yet equally effective, modes of hepatocyte-specific expression.

Phorbol ester modulates interleukin 6- and interleukin 1-regulated expression of acute phase plasma proteins in hepatoma cells.

Interleukin 6 (IL 6) and interleukin 1 (IL-1) regulate the expression of acute phase plasma proteins in rat and human hepatoma cells. Phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), partially mimics the stimulatory effect of IL-6 but reduces that effect of IL-1. TPA and IL-6 act synergistically. These regulatory properties of TPA are also manifested in HepG2 cells transiently transfected with an indicator gene construct carrying the IL-1/IL-6 regulatory enhancer element of the rat alpha 1-acid glycoprotein gene. IL-6 and IL-1 act independently of TPA-inducible kinase C, and of changes in intracellular Ca2+ concentrations. However, prolonged pretreatment of HepG2 cells with TPA results in a drastically reduced cytokine response that is proportional to the loss of cell surface binding activity for the cytokine. These data suggest that hormones activating protein kinase C probably play a contributing role in stimulating the expression of acute phase plasma protein genes but they may be crucial in controlling the responsiveness of liver cells to inflammatory cytokines during subsequent stages of the hepatic acute phase reaction.

Tissue- and species-specific regulation of murine alpha 1-antitrypsin gene transcription.

Previous studies from our laboratories have shown that the tissue specificity of alpha 1-antitrypsin (AT) expression differs among closely related mouse species. In laboratory mice (Mus domesticus), AT mRNA is found almost exclusively in the liver. In the wild-derived species Mus caroli, the mRNA is expressed not only in the liver but also in the kidney, where it is regulated by androgens during post-natal development. We presently show that the tissue specificity, the species specificity, and the developmental regulation of AT mRNA levels correlate with the transcription rate of the AT gene, as measured by nuclear run-on assays. During the course of these experiments, we found that some AT-specific probes are complementary to constitutively synthesized RNAs that do not accumulate and that are unrelated to functional AT mRNA expression. These RNAs, which result from both sense and anti-sense transcription, may derive from aberrant initiation events within certain regions of the AT gene.

Developmental expression, cellular localization, and testosterone regulation of alpha 1-antitrypsin in Mus caroli kidney.

alpha 1-Antitrypsin (alpha 1-protease inhibitor), an essential plasma protein, is synthesized predominantly in the liver of all mammals. We have previously shown that Mus caroli, a Southeast Asian mouse species is exceptional in that it expresses abundantly alpha 1-antitrypsin mRNA and polypeptide, in the kidney as well as the liver (Berger, F.G., and Baumann, H. (1985) J. Biol. Chem. 260, 1160-1165) providing a unique model for examination of the evolution of genetic determinants of tissue-specific gene expression. In the present paper, we have further characterized alpha 1-antitrypsin expression in M. caroli. The extrahepatic expression of alpha 1-antitrypsin is limited to the kidney, specifically within a subset of the proximal tubule cells. The developmental pattern of alpha 1-antitrypsin mRNA expression in the kidney differs from that in the liver. In the kidney, alpha 1-antitrypsin mRNA is present at only 2-4% adult level at birth and increases very rapidly to adult level during puberty between 26 and 36 days of age. There are no significant changes in liver alpha 1-antitrypsin mRNA levels during this period. Testosterone, while having only modest affects on alpha 1-antitrypsin mRNA accumulation in the adult kidney, causes a 20-fold induction of the mRNA in the pre-pubertal kidney. This suggests that the increase in alpha 1-antitrypsin mRNA expression during puberty is testosterone mediated. Southern blot analyses of Mus domesticus and M. caroli genomic DNA and a cloned M. caroli alpha 1-antitrypsin genomic sequence, indicate that a single alpha 1-antitrypsin gene exists in M. caroli, whereas multiple copies exist in M. domesticus. These data show that the alteration in tissue specificity of alpha 1-antitrypsin mRNA accumulation that has occurred during Mus evolution is associated with distinctive developmental and hormonally regulated expression patterns.

Mouse alpha 1-protease inhibitor is not an acute phase reactant.

Mouse plasma contains two major protease inhibitors, alpha 1-protease inhibitor (alpha 1-PI) and contrapsin, which have high affinity for bovine trypsin. Systemic injury, such as turpentine-induced inflammation, did not change the plasma concentration of alpha 1-PI, but increased that of contrapsin by 50%. The concentration of hepatic alpha 1-PI mRNA was determined by Northern blot hybridization and was not significantly affected by the acute phase reaction. J.M. Frazer, S.A. Nathoo, J. Katz, T.L. Genetta, and T.H. Finley [1985) Arch. Biochem. Biophys. 239, 112-119) have reported a threefold increase of mRNA for the elastase specific alpha 1-PI but this increase was not demonstrated by the present study. The mRNAs for known mouse acute phase plasma proteins were, however, stimulated severalfold by the same treatment. These results indicate that in the mouse, as opposed to human, alpha 1-PI is not an acute phase reactant.