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RESEARCH COMMUNICATION |
a Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, BioCity, FIN-20520 Turku, Finland
b Biocenter Oulu and Department of Biochemistry, University of Oulu, Linnanmaa, FIN-90571 Oulu, Finland
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: EGF • FiRE • migration • syndecan-1 • TGF-
• transcription
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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5ß1, vß6, and vß5 integrins, and various proteases such as plasminogen and matrix metalloproteinases (2). In addition, many proteins such as growth factor receptors (3) and cell surface proteoglycans (4, 5) are up-regulated during these processes. Less knowledge is available about transcription factor induction, but evidence exists for wound edge expression of at least the activator protein 1 (AP-1)3 family members c-Jun and c-Fos (6, 7). However, the mechanisms triggering gene expression in mammals during keratinocyte migration are not well understood. Our knowledge is especially restricted about the mechanisms whereby specific genes are turned on and off when the resting keratinocytes become migratory at the wound edge.
While the peptide growth factors seem to play an essential role in the initiation and control of wound healing and reepithelialization by regulating the chemotaxis, proliferation, and migration of different cells (8), they are also major candidates for regulating wounding induced gene expression. Growth factors involved in the reepithelialization process include, at least, the epidermal growth factor (EGF) family members EGF, transforming growth factor alpha (TGF-), and the fibroblast growth factor (FGF) family members FGF-2 and keratinocyte growth factor (KGF or FGF-7). EGFs are produced by several different cell types and act on epithelial cells, stimulating their migration and cell division (9) Likewise, FGF-2 and KGF, which are produced by fibroblasts, act on keratinocytes (10–12). EGF has been shown to enhance wound healing (13) as well as KGF (14) and FGF-2 (15). Besides growth factors, other extracellular signals, including disruption of cell–cell or cell–matrix contacts and the provisional matrix, might contribute to the initiation of migration, reepithelialization, and activation of gene expression (1, 2, 16).
To study the growth factor-initiated transcriptional regulation in vivo, we have made transgenic mice with a previously characterized far upstream FGF-inducible response element (FiRE) (17) in front of a ß-galactosidase reporter gene. The FiRE element has two binding sites for Fos and Jun complexes, called AP-1—one for USF-1 and one for a putatively novel transcription factor, FIN-1. The AP-1 sites are mandatory for activation of FiRE. However, FiRE also requires the other transcription factors adjacent to AP-1s. In fibroblasts, FiRE is induced only by members of the FGF family but not by, for instance, platelet-derived growth factor or EGF (17). The FiRE has been found on the gene syndecan-1, which is a transmembrane heparan sulfate proteoglycan that is strictly regulated during development and tissue regeneration (18). It is known to be up-regulated in migrating and proliferating keratinocytes during cutaneous wound healing, and also in endothelial cells close to the wound site (4, 5). In this study, we show that FiRE is activated selectively in migrating keratinocytes during the reepithelialization phase of wound healing. We also show that the syndecan-1 proximal promoter (SynPr) is not necessary for wounding induced activation of FiRE. Furthermore, by developing a method to monitor FiRE activity in the presence of inhibitory chemicals in organ culture, we suggest that the induction of FiRE requires the involvement of at least two signaling pathways. As this activation is seen only in regenerating and not in resting skin, FiRE could prove to be a valuable tool for wound healing investigations and might have implications in modifying wound healing by targeting genes into wound sites.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MCA3D cells were cultured in Ham's F-12 medium (Gibco Brl, Paisley, U.K.) supplemented with 10% fetal calf serum (FCS). For growth factor treatment, FCS was removed 2 days before adding growth factors. The plasmid constructs used for MCA3D transfections have been described in detail (17). Briefly, the p-271FiRECAT construct contains the FiRE element and 98 bp proximal promoter fragment of syndecan-1 gene in front of chloramphenicol acetyltransferase (CAT) gene. Stable transfections were made by transfecting simultaneously pMAMNeo plasmid and 10-fold molar excess of CAT reporter plasmids by a calcium phosphate method and selecting cells with 750 µg/ml of G418. Several independent clones were pooled.
Wounding experiments
For wound healing studies, 8- to 12-wk-old mice of both sexes were anesthetized by 2.5% avertin and the hair was cut; full thickness incisional wounds were made on the back or on the tails of the mice with a scalpel, and left unsutured and uncovered. The experimental procedures were approved by the ethics committee of the University of Turku. At specified time points, the mice were killed, the wounded parts were removed, fixed in 4% paraformaldehyde for 30 min, washed three times for 30 min, and stained overnight with 1 mg/ml of 5-bromo-4-cholo-3-indolyl-ß-D-galactopyranoside (X-Gal) at RT (22).
Skin culture and analysis of FiRE activation
For organ culture studies, mice were killed and approximately 1 cm2 of intact skin pieces, after hair removal, were removed with a scalpel. They were allowed to float in cell culture conditions overnight in DMEM supplemented with 10% FCS or 2% CMS (carboxy-methyl-Sephadex eluted FCS) in a 24-well plate. Growth factors and chemicals were added to the culture medium for the next 24 h, followed by fixation, ß-galactosidase staining, and production of histological sections. Inhibitory chemicals were added 0.5 h before growth factors or okadaic acid. Growth factors were purchased from PeproTech (London, U.K.) and other compounds from CalbioChem, Nottingham, U.K. (PD 098059, suramin, genistein, bisindolylmalameide) or Sigma, St. Louis, Mo. (anisomycin, wortmannin, rapamycin). SB 203580 was a kind gift from SmithKline Beecham (Philadelphia, Pa.).
To study the impact of the inhibitory chemicals on FiRE activity, mice were killed and their tails were cut into consecutive pieces of approximately 0.5 cm of length (10–12 pieces were obtained from a single tail). Each tail piece was placed separately in a single well of a 24-well plate (Falcon, Oxnard, Calif.) and maintained overnight in cell culture conditions (DMEM supplemented with 10% FCS). The inhibitory chemicals (at concentrations indicated in the text and in 
Figs. 5 and 7) were added to the culture medium, with a lag period of 0.5 to 4 h after cutting the tail. The next day medium was removed, followed by fixation and X-Gal staining, as described above. The keratinocytes tend to migrate over the surface of the cut tail end. These cut ends were photographed with a microscope; the photographs were subsequently scanned (Agfa Arcusscan II) at 150 dots per inch and imported into an image analyzer system of the Microcomputer Image Device system (MCID, Imaging Research Inc.). FiRE activation was evaluated as the area of ß-galactosidase staining (blue color, identified by the analyzing program) proportioned to the total surface of the cut tail end. In each experiment, ß-galactosidase activity in the chemical-treated wound edges was compared to the nontreated (control) tail ends by assigning a value of 1.00 (100%) to the proportional area of the blue color in the control tail ( Fig. 5D and Fig. 7A).
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Immunohistochemistry
Tissues were fixed in 4% formalin, dehydrated in ascending concentrations of ethanol, and embedded in paraffin. Tail pieces were treated with 4% formic acid for 2–4 days before embedment. Microtome sections (5 µM) were mounted on glass slides treated with poly-L-lysine. Standard techniques were used for hematoxylin-eosin staining. For immunohistochemistry, proliferating cell nuclear antigen (PCNA) (Novocastra) recognizing primary antibody and subsequent avidin-biotin-peroxidase complex techniques (Vectastain, Vector Laboratories, Burlingame, Calif.) were used.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fig. 1A).
Because FiRE was known to be growth factor dependent in fibroblasts and because several growth factors are known to participate in wound healing, we targeted our investigations to wounded skin, where syndecan-1 expression is also known to be up-regulated (4, 5). Linear wounds were made to the back and to the tail of mice; the skin and tail were subsequently removed and stained with X-Gal. Dermal cells did not show Lac-Z activity, whereas in all 12 mouse lines that were DNA positive with the FiRE construct, Lac-Z activity was found at the wound site keratinocytes ( Fig. 1B; Fig. 2,
Fig. 3),
in contrast to the six DNA-positive SynPr mouse lines, which illustrated no staining ( Fig. 1C). The intact skin and several other adult organs showed no consistent staining in either SynPr or FiRE strains. However, during embryonic development both FiRE and SynPr strains showed nonspecific Lac-Z activity in neural tissue and limbs, demonstrating that the quiescence of the SynPr strain during wound healing was not due to its chromosomal inactivation. Furthermore, the 2.2 kb proximal promoter fragment has been shown to drive high transcriptional activity in cultured keratinocytes and fibroblasts (19).
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Several time points spanning from 4 h up to 15 days were selected to study the timing of FiRE activation. During the early inflammatory phase (from 4 to 18 h), no Lac-Z activity was seen. However, at 20 to 24 h postwounding, ß-galactosidase staining, indicating activation of FiRE, appeared at the wound edge. This correlates with the time at which keratinocyte migration is assumed to begin. The staining was most abundant at the third day (
Fig. 1B) and started to decline at approximately day 7. To reveal the FiRE activation on different stages of wound healing, consecutive sections of a single tail wound were produced ( Fig. 2). In this model, a cross section from the middle of the incision shows a situation where the keratinocyte migration is just starting, whereas sections from the ends of the incision present a reepithelialized keratinocyte sheet. Staining was first seen in the stratified epidermal sheet of the keratinocytes nearest to the incision site starting migration downward ( Fig. 2A). In the more advanced phase, FiRE activity was very strong at the leading edge of the migratory keratinocytes and in the merging epithelium ( Fig. 2B). During subsequent stratification of epithelial cells, FiRE activation was still evident ( Fig. 2C), but at the end of reepithelialization the staining started to decline, as demonstrated by the most distal section of the tail wound ( Fig. 2D). The epidermis further from the incision site, as well as the dermal cells and the wound clot, remained negative for ß-galactosidase activity throughout the repair process. During the later phases, when dermal integrity was being reestablished and the epidermis still maintained a hyperproliferative state, FiRE was not reactivated. In the hair follicle keratinocytes adjacent to the incision site, FiRE was also activated ( Fig. 2B, arrow). These cells, as well as the wound edge keratinocytes, are supposed to be starting sources for keratinocyte migration.
FiRE is activated in migrating but not in dividing keratinocytes
The relationship between FiRE activity and cell proliferation was investigated by staining the FiRE wound sections with a PCNA antibody (25). During the early time points (e.g., at day 1, when FiRE activity appears at the wound margin), PCNA staining was found to locate more distally, either in the basal keratinocytes and the hair follicle keratinocytes ( Fig. 3A, arrow; Fig. 3C) or in the glandular epithelium. At a more advanced migratory phase (day 2), cell proliferation was more marked in the basal layer, yet in a totally different cell pool than FiRE activity (Fig . 3B). In the fully reepithelialized wound at day 7, where robust cell division was found in the basal keratinocytes, and in the stratum spinosum of the thickened epidermis, the ß-galactosidase staining was detected in only a few cells of the differentiated granular layer ( Fig. 3D). This suggested that the activation of FiRE is strictly restricted to the migrating keratinocytes during wound healing.
Syndecan-1 proximal promoter is not required for activation of FiRE
To investigate the impact of SynPr on the wounding induced activation of FiRE, we constructed an Lac-Z expression plasmid where the 2.2 kb syndecan-1 promoter in the FiRE plasmid was replaced by a 170 bp fragment of the FiRE-TK ( Fig. 4A,
and Materials and Methods). Four independent DNA-positive transgenic mouse strains were obtained by microinjection. Linear wounds were made into the backs and tail of the mice. As with the FiRE-SynPr construct, the FiRE-TK construct was found to be activated at the wound sites in all four strains ( Fig. 4B). Strong ß-galactosidase staining was seen at the migrating keratinocyte sheet while the intact skin as well as the dermis remained negative for Lac-Z activity. Similar to the FiRE-SynPr, the FiRE-TK was also activated in the nearby wound site hair follicle keratinocytes. This demonstrated that FiRE does not require the syndecan-1 promoter to be properly activated during wound healing.
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Activation of FiRE is inhibited by suramin and genistein
FiRE has previously been shown to be activated by FGF-1 and -2 in mesenchymal cells (17). Therefore, we hypothesized that the activation of FiRE would involve growth factors in vivo. To assess the molecular mechanisms inducing FiRE in wounded epithelium, we tested chemicals known to inhibit activation of cell surface receptor tyrosine kinases (RTKs) by growth factors in an organ culture model described in Materials and Methods. It is known that extracted skin pieces are viable for several days in cell culture conditions (26).
Suramin is a well-characterized heparin analog that blocks the binding of several growth factors (e.g., EGF and KGF) (3) to their receptors. It has been shown to have some retarding effects on wound healing without inhibiting reepithelialization (27). Gen~istein, on the other hand, is a specific inhibitor of tyrosine kinases that, among other tyrosine kinases, blocks the growth factor receptors. The ability of these chemicals to inhibit FiRE activity was assayed in a model where mice tails were cut into consecutive pieces after culturing with or without the inhibitors and subsequently stained with X-Gal. Inhibition of the ß-galactosidase activity in the healing surface of the cut tail ends was easily visible ( Fig. 5).
Numerical data were obtained by developing a method where FiRE activation was evaluated as a proportional inten~sity of the Lac-Z activity to the healing surface of the tail pieces (for detailed description of the procedure, please see Materials and Methods). Both suramin and genistein inhibited FiRE activation in a concentration-dependent manner ( Fig. 5). Suramin (100 µM) reduced the staining intensity to approximately 30%; with 300 µM suramin, the activation of FiRE was almost completely blocked ( Fig. 5B, D). The tyrosine kinase inhibitor genistein was also found to inhibit FiRE at 30 µM. The enzyme activity was reduced to approximately 10% at 75 µM and was totally abolished at 150 µM ( Fig. 5C, D). In contrast to growth factor receptor and tyrosine kinase blocking agents, the tyrosine phosphatase inhibitor vanadate had only a marginal effect on FiRE activity at 100 µM ( Fig. 5D). As many of the chemicals are known to be cytotoxic, we investigated whether the lack of FiRE activation is due to total blockage of cell migration. As judged by histological sections, none of the chemicals completely prevented keratinocyte migration ( Fig. 5, lower panels). These data suggested that activation of FiRE in migrating keratinocytes might be dependent on growth factor-induced RTK activation.
Growth factors, together with anisomycin, activate FiRE in nonwounded skin
Since inhibition of growth factor action could block the wounding induced FiRE activity, we tested whether the growth factors could elicit activation of FiRE in intact skin. As introducing various growth factors into the media of cultured nonwounded skins did not activate FiRE, we considered the possibility of a requirement for signaling cascades other than those triggered by growth factors. The Raf/MAPKK1/ERK (Raf/mitogen-activated protein kinase kinase/extracellular regulated kinase) cascade is one well-established pathway known to be induced by growth factors. Therefore, we also stimulated the stress-activated JNK/SAPK and p38 MAPK signaling pathways by anisomycin and UV irradiation. Pieces of skin were tape stripped, which is known to induce at least the EGF receptor (28), and treated with a combination of growth factors (a cocktail of EGF and TGF-) and anisomycin in organ culture. FiRE was not activated if anisomycin ( Fig. 6A)
or growth factors (not shown) were added separately to the culture medium. However, treatment of the skin simultaneously with the growth factors and anisomycin resulted in the activation of FiRE in the keratinocytes of nonwounded skin ( Fig. 6B). Equivalent results were obtained by treating the skin pieces simultaneously with UV irradiation (80 J / m2) and growth factors. Okadaic acid, known to activate several intracellular kinases, Fos and Jun proteins, and AP-1-driven promoters (29) by inhibiting protein phosphatases 1 and 2A, alone was a potent activator of FiRE in resting skin ( Fig. 7B).
The skin of SynPr strains was not in~ducible by any of these treatments (not shown). We also examined whether TGF-, as well as EGF, could elicit activation of FiRE in cultured keratinocytes. Therefore, an immortalized keratinocyte cell line, MCA3D (30), was stably transfected by p-271FiRECAT, a plasmid that contains the FiRE element and 98 bp of syndecan proximal promoter in front of a CAT reporter gene. The transfected cells were grown without serum for 2 days and treated overnight with or without the growth factors, followed by determination of CAT enzyme activity. As shown in Fig. 6C, both EGF and TGF- were able to increase CAT activity by severalfold, indicating that FiRE responds to these growth factors. A reporter plasmid with only the syndecan proximal promoter did not respond to either EGF or TGF- (not shown). All these results suggested that the activation of FiRE can be elicited by growth factors in keratinocytes, but also that the activation might depend on two separate signaling pathways: 1) growth factor-induced RTKs and 2) a stress-induced pathway.
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Inhibiting either ERK or p38 MAP kinases blocks FiRE activity
To further investigate the signal transduction pathways downstream from the cell surface, we used well-characterized compounds known to specifically inhibit different intracellular kinases in an experiment comparable to that shown in Fig. 5( Fig. 7A). PD 098059, a chemical that inhibits activation of MAP kinases ERK-1 and -2 by MAPKK1 at micromolar concentrations (31), was able to reduce activation of FiRE at 5 µM and totally block it at 20 µM, implying involvement of MAP kinases in this activation. A specific inhibitor of stress-induced p38/RK MAPK, SB 203580, shown to be selective for p38 MAPK at least up to 100 µM (32), also acted as an inhibitor of FiRE in a concentration-dependent manner. The ß-galactosidase staining was clearly reduced at 10 µM and abolished at 40 µM concentrations. In contrast to these two compounds, neither the phosphatidylinositol 3-kinase (PI-3 kinase) inhibitor wortmannin (33), even at a concentration of 2000 nM, nor the p70 S6 kinase inhibitor rapamycin, at 100 nM, had any effect on the wound-induced activation of FiRE. Likewise, the protein kinase C (PKC) -specific inhibitor bisindolylmalameide (1.2 µM) failed to inhibit FiRE activation ( Fig. 7A). As well as an inhibitory effect on wounding induced Lac-Z activity, pretreatment with either PD 098059 or SB 203580 was able to abolish okadaic acid ( Fig. 7C, D, respectively), whereas wortmannin up to 5 µM had no inhibitory effect ( Fig. 7E). These results suggest that although the ERK and p38 pathways might be mandatory for transcriptional activation of AP-1-regulated genes in migrating keratinocytes, the PI-3 kinase or the typical PKCs may not be necessary.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Syndecan-1 mRNA and protein levels are induced from 10- to 20-fold in migrating keratinocytes at skin wound sites of adult mice (5). Syndecan-1 is also induced in murine and human endothelial cells, granulation tissue, and dermis (4, 5). Since the activation of FiRE is seen in the migrating keratinocytes, it might be responsible for the induced syndecan-1 expression in these cells. However, in other cells expressing syndecan-1 after wounding, no FiRE activity is seen. This could be due to other elements regulating syndecan-1 expression, but could also indicate that FiRE-induced ß-galactosidase activity is not strong enough to be detected by X-Gal staining in these cells.
Besides syndecan-1, various AP-1-regulated genes including matrix remodeling metalloproteinases (35) and keratins (36) are expressed during trauma and wound healing. Transcription factors Fos and Jun have been proposed to be activated during wounding or wounding induced tumorigenesis (6, 7), and it is likely that Fos and Jun could mediate the growth factor-induced expression of several genes during keratinocyte migration. However, the induction of AP-1 might not to be sufficient to initiate transcription in some promoters, and there could be a requirement for other transcription factors in order to trigger strong expression and enhance the specificity of expression. This seems to be true for activation of FiRE, which is inactivated by deletion of binding sites for transcription factors other than AP-1 in, for example, NIH3T3 cells (17).
The substantial time (at least several hours) required to initiate FiRE activation and keratinocyte migration might be due to earlier signaling events necessary to induce all the mandatory cell surface components on the migratory cells. This could represent, for example, a growth factor-regulated cascade involving several steps, ending in the autocrine release of growth factors and activation of their cognate receptors on migrating keratinocytes. For example, KGF is released by fibroblasts and acts on keratinocytes, where it has been shown to induce the expression of TGF- and EGF receptor (EGFR) (37). Activation of FiRE could be delayed until sufficient amounts of EGFR are available at the cell surface. EGF and TGF- are known to induce proliferation of keratinocytes (9, 11), whereas the dividing keratinocyte population is completely FiRE negative. This might be due to the lack of activated stress receptors or maintained cell–cell contacts. On the other hand, FiRE-negative dermal cells might also lack several components demanded for activation, including growth factor receptors such as EGFR, insufficient activation of stress-induced signals, and activation of required transcription factors. The attenuation of FiRE after reepithelialization could be due not only to the down-regulation of local growth factor release, but also to the down-regulation of stress-activated signals after reestablishing cell-to-cell and cell-to-matrix contacts.
In addition to RTK-activating growth factors such as EGF, which are known to regulate the ERK signaling cascade, the activation of FiRE is dependent on other extracellular stimuli, triggering the stress-activated p38 MAPK pathway. In cultured MCA3D keratinocytes, FiRE could be activated only by growth factor treatment, which might be due to the constitutive activity of p38 MAPK in these cells (P. Jaakkola, unpublished observation). Several different cellular stresses, such as UV irradiation and osmotic shock, have been shown to activate p38, though they are poor activators of ERKs (38). Besides the diverse stresses, some cytokines (including interleukins and TNF-) are able to induce p38 (39). The requirement of UV irradiation or anisomycin, together with growth factors of the EGF family, implies that the growth factors are not responsible for activation of p38, whereas the growth factors are prime candidates for triggering signals of FiRE activation via ERK. What the signal (or signals) is that triggers the p38 in migrating keratinocytes during wound healing remains to be investigated. Several possibilities exist, including involvement of interleukins, the local release of cell degradation products, or the loss of cell to matrix contacts and subsequent changes in adhesion molecule expression. It has been suggested that both the growth factor and stress-induced activations could be triggered through RTKs (40). Although the growth factor receptors can also be the major starting point for stress-induced signaling pathway in some biological situations, in our model there is a requirement of both growth factor- and stress-regulated events to activate FiRE in nonwounded skin, which suggests that these stimuli do not trigger the same kinase activity of the receptors or subsequent downstream mediators. However, whether the two stimuli might still be triggered by different growth factor receptors or by activating different subset of signaling molecules by one type of growth factor receptor cannot be ruled out. Whatever the explanation is, it is implied that gene transcription requires a variety of signals during wound reepithelialization.
FiRE can be considered a healing process-related gene response element. Our initial analysis of the human homologue of FiRE has indicated a high similarity between the human and murine DNA sequences (J.-P. Pursiheimo et al., unpublished observation). Whether it is also activated in the context of other growth factor-regulated regeneration processes remains to be studied. Although found upstream from the gene of syndecan-1, the FiRE or similar elements might also function to up-regulate other genes in reepithelializating wound keratinocytes. This is suggested by the fact that FiRE is activated at wound sites regardless of the basal promoter it cooperates with. FiRE will be a highly useful tool for growth factor-induced and healing-related gene expression studies, and it might also have therapeutic applications, for example, in poorly healing ulcerative wounds where FiRE-driven therapeutic agents, such as keratinocyte and fibroblast chemoattractants or mitogens, could be introduced into wound margin keratinocytes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Correspondence: Turku Centre for Biotechnology, Tykistökatu 6B, BioCity, FIN-20520 Turku, Finland. E-mail: markku.jalkanen{at}btk.utu.fi
3 Abbreviations: AP-1, activator protein 1; CAT, chloramphenicol acetyltransferase; CMS, carboxy-methyl-Sephadex eluted FCS; EGF, epidermal growth factor; ERK, extracellular regulated kinase; FGF-2, basic fibroblast growth factor; FiRE, FGF-inducible response element FCS, fetal calf serum; KGF, keratinocyte growth factor; MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; PCNA, proliferating cell nuclear antigen; PKC, protein kinase C; PI-3 kinase, phosphatidylinositol 3-kinase; RTK, receptor tyrosine kinase; TGF-, transforming growth factor alpha; X-Gal, 5-bromo-4-cholo-3-indolyl-ß-D-galactopyranoside; SynPr, syndecan-1 proximal promoter; FiRE-TK, FiRE-thymidine kinase promoter.
Received for publication December 22, 1997. Accepted for publication February 23, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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