A crystallinity study of dental tissues and tartar by infrared spectroscopy.

In this paper we report a study of an important property of biomineralized phases, crystallinity, on the basis of previous results for synthetic apatite. Crystallinity is not only important for understanding biomineralization, it is also related to the maturation and mechanisms of growth of calcium phosphates in biological surroundings. We studied two kinds of sample, teeth as an example of biomineralized tissues and dental calculi (adhering) as an example of mineralization without participation of biological agents, except possibly bacteria. The investigation focused on study of ν(1)-ν(3) infrared absorption bands of PO(4)(3-) phosphates. We used ATR (attenuated total reflection) analysis to examine human dental tissues and tartar on several samples. The results confirm for the first time previous assumptions about the growth and maturation of dental calculi, i.e., crystallinity progresses from regions of high crystallinity to regions of lower crystallinity, and, in addition, its quantification with spatial resolution in the sample. A gradual pattern was observed in dental calculus. Another result from this study was that cementum and dentine had similar crystallinity, despite their different biological and mechanical functions.

Heparin-binding EGF-like growth factor stimulation of smooth muscle cell migration: dependence on interactions with cell surface heparan sulfate.

Heparin-binding EGF-like growth factor (HB-EGF), but not EGF, binds to cell surface heparan sulfate proteoglycan (HSPG). This was demonstrated in (a) the binding of 125I-HB-EGF to mutant CHO cells deficient in HS production was diminished by 70% compared to wild-type CHO cells, (b) the binding of 125I-HB-EGF to CHO cells and bovine aortic smooth muscle cells (BASMC) was diminished 80% by heparitinase or chlorate treatment, and (c) 125I-EGF did not bind to CHO cells and its binding to BASMC was not diminished at all by heparitinase and only slightly by chlorate treatment. Accordingly, the role of HB-EGF interactions with HSPG in modulating bioactivity was examined. Heparitinase or chlorate treatment of BASMC diminished the ability of HB-EGF to stimulate BASMC migration by 60-80%. A similar inhibition of migration occurred when BASMC were treated with a synthetic peptide (P21) corresponding to the sequence of the putative heparin-binding domain of HB-EGF. As a control for BASMC viability, and for specificity, it was found that heparitinase and P21 did not inhibit at all and chlorate inhibited only slightly the stimulation of BASMC migration by PDGF AB. Since heparitinase, chlorate, and P21 treatment also diminished by 70-80% the cross-linking of 125I-HB-EGF to the EGF receptor, it was concluded that the interaction of HB-EGF, via its heparin-binding domain, with cell surface HSPG was essential for its optimal binding to the EGF receptor on BASMC and hence for its optimal ability to stimulate migration.

The presence of fibroblast growth factor in the frog egg: its role as a natural mesoderm inducer.

A complementary DNA clone corresponding to a 4.2-kilobase transcript that is present in the Xenopus oocyte and newly transcribed in the neurula stages of development has been isolated. This messenger RNA encodes a 155-amino acid protein that is 84% identical to the human basic fibroblast growth factor (bFGF). When expressed in Escherichia coli and purified, the Xenopus FGF induced mesoderm in animal cell blastomeres as measured by muscle actin expression. Immunoblots with an antibody to a Xenopus FGF peptide show that the oocyte and early embryo contain a store of the FGF polypeptide at high enough concentrations to induce mesoderm. The presence of FGF in the oocyte, together with the apparent lack of a secretory signal sequence in the protein, suggest that the regulation of mesoderm induction may involve novel mechanisms that occur after the translation of FGF.

A heparin-binding growth factor secreted by macrophage-like cells that is related to EGF.

Macrophage-like U-937 cells secrete a 22-kilodalton heparin-binding growth factor that is mitogenic for BALB-3T3 fibroblasts and smooth muscle cells, but not endothelial cells. The amino acid sequence predicted from complementary DNA clones indicates that the mitogen is a new member of the epidermal growth factor (EGF) family. This heparin-binding EGF-like growth factor (HB-EGF) binds to EGF receptors on A-431 epidermoid carcinoma cells and smooth muscle cells, but is a far more potent mitogen for smooth muscle cells than is EGF. HB-EGF is also expressed in cultured human macrophages and may be involved in macrophage-mediated cellular proliferation.

Nucleotide sequence of a bovine clone encoding the angiogenic protein, basic fibroblast growth factor.

Basic and acidic fibroblast growth factors (FGF's) are potent mitogens for capillary endothelial cells in vitro, stimulate angiogenesis in vivo, and may participate in tissue repair. An oligonucleotide probe for bovine basic FGF was designed from the nucleotide sequence of the amino-terminal exon of bovine acidic FGF, taking into account the 55 percent amino acid sequence homology between the two factors. With this oligonucleotide probe, a full length complementary DNA for basic FGF was isolated from bovine pituitary. Basic FGF in bovine hypothalamus was shown to be encoded by a single 5.0-kilobase messenger RNA; in a human hepatoma cell line, both 4.6- and 2.2-kilobase basic FGF messenger RNA's were present. Both growth factors seem to be synthesized with short amino-terminal extensions that are not found on the isolated forms for which the amino acid sequences have been determined. Neither basic nor acidic FGF has a classic signal peptide.

Involvement of wound-associated factors in rat brain astrocyte migratory response to axonal injury: in vitro simulation.

The poor ability of mammalian central nervous system (CNS) axons to regenerate has been attributed, in part, to astrocyte behavior after axonal injury. This behavior is manifested by the limited ability of astrocytes to migrate and thus repopulate the injury site. Here, the migratory behavior of astrocytes in response to injury of CNS axons in vivo was simulated in vitro using a scratch-wounded astrocytic monolayer and soluble substances derived from injured rat optic nerves. The soluble substances, applied to the scratch-wounded astrocytes, blocked their migration whereas some known wound-associated factors such as transforming growth factor-beta 1 (TGF-beta 1), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), and heparin-binding epidermal growth factor in combination with insulin-like growth factor-1 (HB-EGF + IGF-1) stimulated intensive migration with consequent closure of the wound. Migration was not dominated by proliferating cells. Both bFGF and HB-EGF + IGF-1, but not TGF-beta 1, could overcome the blocking effect of the optic nerve-derived substances on astrocyte migration. The induced migration appeared to involve proteoglycans. It is suggestive that appropriate choice of growth factors at the appropriate postinjury period may compensate for the endogenous deficiency in glial supportive factors and/or presence of glial inhibitory factors in the CNS.

Production of heparin binding epidermal growth factor-like growth factor in the early phase of regeneration after acute renal injury. Isolation and localization of bioactive molecules.

We have recently reported that heparin-binding epidermal growth factor-like growth factor (HB-EGF) mRNA is induced in the rat kidney after acute ischemic injury. The present studies were designed to investigate whether bioactive HB-EGF protein is also produced in response to renal injury induced by either ischemia/reperfusion or aminoglycosides. Heparin-binding proteins were purified from kidney homogenates by heparin affinity column chromatography using elution with a 0.2-2.0 M gradient of NaCl. A single peak of proteins that eluted at 1.0-1.2 M NaCl was detected in the postischemic kidney within 6 h of injury. This eluate fraction stimulated DNA synthesis in quiescent Balb/c3T3, RIE, and NRK-52E cell lines, all of which are responsive to the epidermal growth factor family of mitogenic proteins. The EGF receptor of A431 cells was also tyrosine phosphorylated by this eluate peak. Furthermore, immunoblotting with a polyclonal antibody against rat HB-EGF indicated that the eluate peak contained immunoreactive proteins of 22 and 29 kD mol wt, consistent with the reported sizes of the secreted form and membrane anchored form of HB-EGF, respectively. Immunohistochemical studies revealed that HB-EGF was produced predominantly in distal tubules in kidneys injured either by ischemia/reperfusion or aminoglycoside administration. We also found that during metanephric development immunoreactive HB-EGF was detected in the ureteric bud as early as E14.5 and persisted in structures arising from the ureteric bud throughout embryogenesis. These results suggest that in response to acute injury, HB-EGF is produced predominantly in distal tubules and that endogenous HB-EGF may be an important growth factor involved in renal epithelial cell repair, proliferation, and regeneration in the early stages of recovery after acute renal injury, as well as in nephrogenesis.

Heparin-binding EGF-like growth factor contributes to reduced glomerular filtration rate during glomerulonephritis in rats.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the epidermal growth factor (EGF) family, is expressed during inflammatory and pathological conditions. We have cloned the rat HB-EGF and followed the expression of HB-EGF in rat kidneys treated with anti- glomerular basement membrane (anti-GBM) antibody (Ab) to induce glomerulonephritis (GN). We observed glomerular HB-EGF mRNA and protein within 30 minutes of Ab administration and showed by in situ hybridization that glomerular HB-EGF mRNA expression was predominantly in mesangial and epithelial cells. Expression of HB-EGF correlated with the onset of decreased renal function in this model. To test the direct effect of HB-EGF on renal function, we infused the renal cortex with active rHB-EGF, prepared from transfected Drosophila melanogaster cells. This treatment induced a significant decrease in single nephron GFR (SNGFR), single nephron plasma flow, and glomerular ultrafiltration coefficient and an increase in the glomerular capillary hydrostatic pressure gradient. In addition, anti-HB-EGF Ab administered just before anti-GBM Ab blocked the fall in SNGFR and GFR at 90 minutes without any change in the glomerular histologic response. These studies suggest that HB-EGF expressed early in the anti-GBM Ab GN model contributes to the observed acute glomerular hemodynamic alterations.

Addition of growth hormone secretion signal to basic fibroblast growth factor results in cell transformation and secretion of aberrant forms of the protein.

Basic fibroblast growth factor (bFGF) is a potent mitogen for a wide variety of cell types. Unlike most growth factors, the primary translation product for bFGF appears to lack a secretory signal peptide. To explore the normal mode of bFGF release, as well as to investigate the growth factor's oncogenic potential, expression vectors were created for a bFGF cDNA and for a chimeric molecule in which the bFGF coding sequence was linked to the human growth hormone signal peptide sequence. Transfection of NIH3T3 cells with the bFGF cDNA vectors caused the synthesis of high levels of biologically active, cell-associated bFGF, but no evidence of transformation was detected. In contrast, the chimeric bFGF-signal peptide expression vector induced foci of transformation at a very high frequency. The transformed cells grew in soft agar and were tumorigenic in nude mice. The majority of the immunoreactive bFGF species made by the transformed cells was found in the conditioned medium and appeared to be posttranslationally modified, indicating that the chimeric bFGF-signal peptide molecule was processed through the secretory pathway. The secreted bFGF exhibited little mitogenic activity, suggesting that interaction of bFGF with its receptor likely occurs while the fusion protein is being processed along the secretory pathway.

Basic fibroblast growth factor stimulation of epidermal wound healing in pigs.

Basic fibroblast growth factor (bFGF) has recently been shown to be a mitogen for keratinocytes. This observation has now been extended in a porcine model of epidermal wound healing. A single application of recombinant human bFGF given at the time of injury to healthy animals accelerated the rate of epithelialization by 20%; multiple applications gave no greater effect than the single application. Histologic analysis of biopsies of these partial-thickness wounds taken during bFGF-mediated healing supported the assessment of an enhanced rate of epithelialization and an earlier onset of dermal healing. Because no histologic abnormalities were observed, bFGF induced an acceleration of what appears to be the normal healing process.

Differential regulation of heparin-binding epidermal growth factor-like growth factor in the adult ovariectomized mouse uterus by progesterone and estrogen.

Expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) was studied in the adult ovariectomized mouse uterus in response to progesterone (P4) and/or 17 beta-estradiol (E2) using Northern blotting, in situ hybridization, and immunohistochemistry. A 2.5-kilobase transcript of HB-EGF messenger RNA (mRNA) was detected in total uterine RNA samples. Although low levels of this mRNA were detected in uterine samples of oil-treated ovariectomized mice (control), an injection of E2 promptly up-regulated the levels. The mRNA levels peaked at 2 h and returned to basal levels after 12 h. Injection of P4 alone did not influence the basal levels; however, coinjection of E2 with P4 caused a rapid, but transient, up-regulation of the mRNA. The levels peaked between 2-4 h and declined 6 h after the hormone injections. Coinjection of E2 with P4 after 1 day of P4 priming also resulted in peak levels of HB-EGF mRNA at 2 h; however, the levels were not sustained thereafter. Because P4 and E2 differentially regulate heterogeneous uterine cell types, in situ hybridization was performed to determine cell-specific expression of HB-EGF mRNA in the ovariectomized uterus before and after steroid treatments. In the oil-treated uterine sections, very low levels of autoradiographic signals were observed in the luminal epithelium. In contrast, an injection of E2 resulted in a marked accumulation of HB-EGF mRNA primarily in uterine epithelial cells within 2 h. Although specific hybridization signals could not be detected in any uterine cell types after P4 treatment, combined treatment with P4 and E2 resulted in an accumulation of HB-EGF mRNA in stromal cells. To determine whether uterine HB-EGF mRNA was translated, cellular distribution of HB-EGF protein was investigated by immunohistochemistry. In oil-treated uterine sections, an overall weak immunostaining was noted, whereas no staining could be detected in uterine sections after P4 treatment. In contrast, positive immunostaining was noted in epithelial cells after E2 treatment. Coinjection of P4 with E2 caused immunostaining in the stroma. These results are consistent with those of in situ hybridization. The present investigation establishes that in the adult ovariectomized mouse uterus, E2 regulates HB-EGF expression in the epithelium, whereas expression of HB-EGF in the stroma is regulated by P4 and E2.

Basic fibroblast growth factor: production and growth stimulation in cultured adrenal cortex cells.

Cultured bovine adrenal cortex cells express the basic fibroblast growth factor (bFGF) gene and contain, but under normal conditions apparently do not release, bFGF. However, once released, bFGF can stimulate proliferation of the cells, indicating that it could act as a self-stimulating growth factor for adrenal cortex cells. It is conceivable that the intracellular bFGF is released upon injury of the adrenal cortex and that it may be involved in the subsequent tissue repair mechanisms by stimulating the proliferation of adrenal cortical and vascular endothelial cells.

Insulin-like growth factor-binding protein-3 induces fetalization in neonatal rat cardiomyocytes.

We employed cDNA microarrays representing 4000 distinct sequences to profile changes in gene expression in a rodent model of heart disease, namely, progression to heart failure after myocardial infarction. Differential gene expression in the left ventricle was examined at 4-week intervals over a 12-week period after coronary artery ligation in rats. Over this time course, insulin-like growth factor-binding protein-3 (IGFBP-3) was found to have a greater expression than in nondiseased tissues. We then employed quantitative real-time PCR to analyze gene expression in neonatal rat cardiac myocytes that had been treated with recombinantly expressed IGFBP-3 to examine a number of transcriptional responses designed to reflect the heart failure phenotype. The IGFBP-3 protein was shown to induce transcription of atrial natriuretic factor (ANF) and beta-myosin heavy chain (B-MHC). Analysis of conditioned media taken from IGFBP-3-treated cardiac myocyte cultures demonstrated an increase in ANF protein as well as in protein synthesis, as determined by metabolic incorporation of a radiolabeled amino acid. However, transcriptional changes of troponin-1, endothelin-1, or angiotensin-II by IGFBP-3 were not observed.

Involvement of double-strand chromosomal breaks for mating-type switching in Saccharomyces cerevisiae.

The yeast S. cerevisiae switches a and alpha cell types by a transposition mechanism that replaces genetic information residing at the mating-type locus (MAT) with information copied from either of the two donor loci, HML and HMR. The donor HML and HMR loci contain the same genetic information as the MATa and MAT alpha alleles, yet they do not switch. Additionally, Strathern et al. (1982) have described an in vivo double-strand DNA break found at subgenomic levels (approximately 2% of MAT DNA) within the MAT locus but not within HML and HMR. We have examined the role of this double-strand DNA break in the switching process. Cell lineage studies show that strains containing deletions of the donor HML and HMR loci produce lethal progeny in the exact pattern described for MAT switching in standard strains. Our interpretation is that the double-strand MAT break in the deletion strains cannot be repaired because of the lack of the donor loci, resulting in cell death. We suggest that the double-strand DNA break is an initiating event for switching and that this event is lethal in the absence of the donor loci. MAT mutants isolated as survivors from this "pedigree of death" define a site required for switching where the double-strand break occurs. We have also examined marl mutant strains in which the donor loci are expressed and observed to switch (Klar et al. 1981a). The double-strand DNA cut appears at the HM loci in these strains. Thus, there is a strong correlation between the presence or absence of the double-strand break at each cassette and its ability or inability to switch as observed at the single cell level.

Heparin-binding EGF-like growth factor synthesis by smooth muscle cells.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been previously demonstrated to be a potent mitogen for smooth muscle cells. Evidence is now presented that these cells synthesize HB-EGF as well. Cultured fetal human vascular smooth muscle cells express 2.5-kb HB-EGF mRNA. These cells also release an HB-EGF-like activity that (i) stimulates smooth muscle cell and BALB/c 3T3 cell but not endothelial cell proliferation; (ii) binds to TSK heparin affinity columns and is eluted with 0.9-1.2 M NaCl, and (iii) triggers phosphorylation of a protein with the same molecular weight as the 170-kD EGF receptor. In addition, 125I-HB-EGF can be cross-linked to the EGF receptor on fetal human vascular smooth muscle cells. These results suggest that smooth muscle cells can both synthesize and respond to HB-EGF, and that HB-EGF may therefore be involved in autocrine regulation of these cells.

Isolation and characterization of a vascular endothelial cell mitogen produced by pituitary-derived folliculo stellate cells.

A growth factor with specificity for vascular endothelial cells has been identified in conditioned medium of pituitary-derived folliculo stellate cells. This factor, named folliculo stellate-derived growth factor (FSdGF), was purified to homogeneity by a combination of heparin-Sepharose affinity chromatography, Bio-Gel P-60 exclusion chromatography, Mono S ion-exchange chromatography, and hydrophobic chromatography on a C4 reverse-phase HPLC column. FSdGF was characterized as a homodimer composed of two subunits with a molecular mass of 23 kDa. FSdGF was a potent mitogen for vascular endothelial cells with activity detectable at 25 pg/ml and saturation at 500 pg/ml. It did not stimulate the proliferation of other cell types such as bovine vascular smooth muscle cells, corneal endothelial cells, adrenal cortex cells, granulosa cells, BALB/MK cells, or BHK-21 cells. Microsequencing revealed an N-terminal sequence having no significant homology to any known protein. The release of FSdGF by pituitary cells and its target cell specificity raise the possibility that FSdGF may play a role in angiogenesis.

Heparin blockade of thrombin-induced smooth muscle cell migration involves inhibition of epidermal growth factor (EGF) receptor transactivation by heparin-binding EGF-like growth factor.

Agonists of G protein-coupled receptors, such as thrombin, act in part by transactivating the epidermal growth factor (EGF) receptor (EGFR). Although at first a ligand-independent mechanism for EGFR transactivation was postulated, it has recently been shown that this transactivation by various G protein-coupled receptor agonists can involve heparin-binding EGF-like growth factor (HB-EGF). Because thrombin stimulation of vascular smooth muscle cell migration is blocked by heparin and because heparin can displace HB-EGF, we investigated the possibility that thrombin stimulation of smooth muscle cells (SMCs) depends on EGFR activation by HB-EGF. In rat SMCs, EGFR phosphorylation and extracellular signal-regulated kinase (ERK) activation in response to thrombin are inhibited not only by the EGFR inhibitor AG1478 and by EGFR blocking antibody but also by heparin and by neutralizing HB-EGF antibody. HB-EGF-dependent signaling induced by thrombin is inhibited by batimastat, which suggests a requirement for pro-HB-EGF shedding by a metalloproteinase. We further demonstrate that this novel pathway is required for the migration of rat and baboon SMCs in response to thrombin. We conclude from these data that the inhibitory effect of heparin on SMC migration induced by thrombin relies, at least in part, on a blockade of HB-EGF-mediated EGFR transactivation.