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a Centre for Molecular Biotechnology, School of Life Sciences, Queensland University of Technology, Brisbane, Queensland 4001, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-nerve growth factor), Klk9 (epidermal growth factor-binding protein), and Klk21 in the early conceptus (until the 2-cell stage). Only Klk21 continued to be expressed in the blastocyst until Day 7.5 of pregnancy. Expression of Klk9 reappeared at Day 7.5 and was consistently detected until Day 11, the last day studied; Klk1 was again expressed in the embryo from Day 9.5, with decreased levels by Day 11. In contrast, in the uterus or decidua, there was no expression of Klk1 until Day 7.5, when mRNA transcripts were abundant; transcripts then decreased in the Day 9.5 and Day 11 uterus. Expression of Klk21 in the uterus and decidua displayed a similar pattern but was detected at much lower levels. Interestingly, a novel Klk21-like mRNA was also detected in uterine tissue samples but not in embryonic samples; Klk3, Klk5, and Klk9 were not consistently expressed in the uterus or decidua over this time. This is the first report of the expression of specific kallikreins during early development. The distinct gene- and tissue-specific expression patterns presented in this study, in conjunction with the well-characterized roles of kallikreins in regulation of protein activation, ECM degradation, and proliferative events, suggests the involvement of the kallikrein gene family during early development.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, the kallikrein gene family has also been implicated in various events of uterine physiology including embryo implantation [10–14]. The kallikreins encode proteins that activate a wide range of substrates including ECM proteinases and growth factors [15]. Therefore, they have involvement in many biological functions including regulation of local blood flow, angiogenesis, tissue invasion, and mitogenesis [16]—events that, as described above, are integral to early development. For example, tissue kallikrein can activate both MMP-2 and MMP-9 [17, 18]. Other kallikrein members of the human and mouse families have demonstrated activity on pro-uPA [19, 20]. Kallikreins may also be directly involved in trophoblast invasion, as they are able to degrade ECM components such as fibronectin and laminin [21–23].
With respect to growth factor activation, the activation of EGF in the mouse is dependent on an EGF-binding protein (EGF-BP) that is encoded by a kallikrein gene [24, 25]. In addition, a member of the human kallikrein family has been shown to degrade insulin-like growth factor-binding protein-3, thus regulating the bioavailability of IGF-1 [26]. Interestingly, both EGF and IGF-1 have been shown to be expressed from the uterus during implantation and are important factors for normal development [7]. It has been suggested that the proliferative and vascular changes during early development occur in a similar manner to an inflammatory reaction [27]. Tissue kallikrein, via the activation of bradykinin, a potent vasoactive peptide, is a well-characterized mediator in inflammation [15, 16].
Recent studies have shown that immunoreactive kallikrein in the uterus progressively rises toward the peak of invasive activity of the embryo in the rat [12, 28]. Furthermore, this increase is specifically located in the implantation sites, in contrast to interimplantation sites [14, 28]. Interestingly, estrogen and progesterone, which play a prominent role in controlling the expression of the proteinases and growth factors involved in implantation, also regulate kallikrein expression in the human endometrium [11] and during early pregnancy in the rat [13]. Although these data demonstrate the involvement of kallikreins in these uterine events, expression of specific members of this large gene family has not been fully characterized at this time.
The aim of this study was to elucidate the temporal and tissue-specific expression of individual members of the kallikrein family during early murine development, which is the best-characterized model of early development. In a previous study, in which the tissue-specific expression of 12 mouse kallikreins was established, 5 mouse kallikreins (Klk1, Klk3, Klk5, Klk9, and Klk21) were demonstrated to be expressed in the nonpregnant uterus [29] (personal communication with M. Digby). In this study, we have extended these findings to determine the pattern of expression of these five Klk genes at the time of implantation using a sensitive and specific reverse transcription-polymerase chain reaction (RT-PCR) strategy. The specificity of the RT-PCR strategy was supported by DNA sequencing and/or Southern hybridization. Although the functions of Klk5 and Klk21 are not yet characterized, Klk1 is known to encode tissue kallikrein, and Klk3 and Klk9 encode the proteins -nerve growth factor (NGF) and EGF-BP, respectively [25]. As noted above, both tissue kallikrein and EGF have been associated with implantation. An understanding of the tissue-specific and temporal expression of this gene family will contribute to our understanding of the important regulators of this critical event in early development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Six-week-old, superovulated, mated female BCBF1 mice were obtained from Central Animal Breeding House, The University of Queensland. Embryos at the fertilized egg stage, 2-cell stage, and blastocyst stage (Day 1, Day 2, and Day 4 of pregnancy, respectively), as well as Day 11 embryos, were collected between 0900 h and 1100 h. Embryos at Days 7.5 and 9.5 of pregnancy were collected between 1500 and 1700 h. Uterine tissue was collected at corresponding stages (except Day 2) and separated from the decidua, which was collected at Days 7.5 and 9.5 of pregnancy only. Separation of tissues was carried out under low magnification with a dissecting microscope (Leica, MZ6; Milton Keynes, UK). Submandibular salivary glands were also removed for use as positive controls for kallikrein expression. After collection, tissue samples were snap frozen in liquid nitrogen and stored at -80°C. This project was performed in accordance with the National Health and Medical Research Council Code of Practice for Animal Research and was approved by the Queensland University of Technology Animal Ethics Committee.
RNA Extraction and Reverse Transcription (RT)
Total RNA was isolated from the frozen tissues by RNAzol B isopropanol/chloroform extraction (Biotecx Laboratories Inc., Houston, TX) according to the manufacturer's instructions, except that the isopropanol precipitation of RNA was left overnight at -20°C before centrifugation. The final pellet was resuspended in 50 µl diethylpyrocarbonate-treated sterile distilled water. Purity was measured by optical density (260/280-nm ratio) with all samples having ratios greater than 1.8, indicating little or no protein contamination.
Five micrograms of uterine, decidual, or periimplantation embryo RNA was reverse transcribed with oligo(dT) priming. For preimplantation embryos, the total RNA from 60–100 fertilized eggs, 2-cell embryos, or blastocysts was added into the RT reaction. To anneal the oligo(dT) primers to the template, the RNA was incubated with 0.5 µg oligo(dT) primer at 70°C for 10 min. First-strand synthesis was achieved by incubating with 200 U Superscript II in 5-strength buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl2), 4 mM dithiothreitol (all from Gibco BRL Life Technologies, Sydney, Australia), and 0.2 mM dNTPs at 42°C for 90 min in a 20-µl reaction. The reaction was stopped by inactivating the enzyme at 70°C for 10 min and then diluting the reaction to a 50-µl volume.
PCR Amplification with Gene-Specific Primers
Integrity of the cDNA was first assessed by amplifying a region of the mouse ß-actin gene. Amplification with ß-actin-specific primers, which bracket an intron, gave a 243-base pair (bp) product from cDNA and a larger 330-bp product if genomic DNA was present. Samples shown to contain genomic DNA were discarded. Reactions were performed in a PTC-200 DNA Engine (MJ Research, Watertown, MA) with 1 µl of cDNA (equivalent to 0.5 µg uterus, decidua, or periimplantation embryo RNA, or total RNA from 1–2 preimplantation embryos), 20 pmol of each ß-actin-specific primer (forward: 5'-CGT GGG CCG CCC TAG GCA CCA-3'; reverse: 5'-TTG GCC TTA GGG TTC AGG GGG G-3') [30] or the kallikrein-specific primers (see below), 15 nmol dNTPs, 10-strength buffer (100 mM Tris-HCl, 15 mM MgCl2), and 1 U Taq DNA polymerase (all from Roche, Castle Hill, Australia) in a total reaction volume of 50 µl. PCR for ß-actin was performed with 2-min initial denaturation at 94°C and 35 cycles of 94°C for 1 min, annealing at 55°C for 1 min 30 sec, and extension at 72°C for 1 min 30 sec. Annealing temperatures varied slightly for each set of kallikrein primers and ranged from 56° to 58°C. Thirty-five cycles was chosen, as this was within the linear amplification for the Klks (except for Klk3, 40 cycles) and allowed comparative analysis of Klk expression with the ubiquitously expressed ß-actin. Submandibular salivary gland cDNA were used as positive controls for kallikrein expression in all PCR reactions.
Specific forward and reverse primers were designed for each kallikrein, targeted to regions of greater variability and spanning at least one intron. The mouse kallikrein family is the largest described and also the most conserved, with 82–93% homology at the nucleotide level [31] and 70–85% at the amino acid level [15]. In designing gene-specific primers, although sequence similarities with other family members were considered, of paramount importance was the discrimination between those kallikreins (Klk1, Klk3, Klk5, Klk9, and Klk21) that were found to be expressed in the uterus by Digby [29]. Table 1 shows the primers that were designed and their position within the gene. The actual variation between the primer sequence for each gene against those of all other family members is presented in Table 2.
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Primers specific to Klk1 and Klk21 were more difficult to design, since these two kallikreins are particularly conserved. Given this, RT-PCR amplification of Klk1 and Klk21 was conclusively identified by Southern hybridization with internal probes (1P and 21P) that specifically discriminated against those kallikreins that also may have been amplified (see Table 2).
Identification of PCR Products
Ten microliters of PCR product and 4-µl loading buffer were resolved by electrophoresis at 100 V on a 2% agarose gel in TAE buffer (2 M Tris-acetate, 0.5 M EDTA, pH 7.8) and visualized by ethidium bromide staining. Products were then analyzed either by Southern hybridization or by DNA sequencing.
Southern Hybridization
Electrophoresed products were transferred to a Hybond-N+ (Amersham, Arlington Heights, IL) nylon membrane, prehybridized for 2 h at 37°C in digoxigenin (DIG) Easy Hyb (Roche), and then hybridized with 20 pmol of DIG-labeled probe overnight at 37°C. The oligonucleotide probes were end-labeled with terminal transferase and DIG-dTTP as described in the manufacturer's protocol (Roche). Washes were first performed in double-strength SSC/0.1% SDS, 0.5-strength SSC/0.1% SDS, then in 0.1-strength SSC/0.1% SDS (1P probe) or 0.2-strength SSC/0.1% SDS (21P probe) at 37°C (single-strength SSC is 0.15 M sodium chloride and 0.015 M sodium citrate). Hybridization was detected by conjugation with DIG antibody, chemiluminescent detection with CDPstar (Roche), and exposure of the membrane to Agfa Curix RP1 Plus x-ray film (Agfa, Nunawading, Australia) at room temperature.
The stringency of the hybridization and subsequent washes was monitored by dot-blot controls containing PCR-amplified and sequenced Klk1, Klk3, Klk5, Klk9, Klk11, and Klk21 cDNA products.
PCR Product Cloning and DNA Sequencing
Representative PCR products were excised from the agarose gel and purified using the QIAquick Gel Extraction Kit (QIAGEN, Clifton Hill, Australia). Purified PCR product was ligated to an expression vector, pGEM-T Easy (Promega, Annandale, Australia), and transformed. Plasmid DNA from recombinant colonies were purified using a QIAGEN miniprep kit, and the inserts were sequenced using vector primers and the ABI PRISM DyeDeoxy Terminator Cycle Sequencing Kit on an automated Applied Biosystems 373A DNA Sequencer (Applied Biosystems, Scoresby, Australia). Sequences were analyzed using the FastA program of the Australian National Genomic Information Service (Sydney, Australia).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Replicates of embryo, uterus, and decidua samples were collected from different mice at different stages of early development. The amplification of the "housekeeping" gene, ß-actin, which was ubiquitously expressed in all samples (Figs. 1a, 2a, and 3a), confirmed the quality of the cDNA and lack of genomic contamination.
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Identification of Kallikrein Expression
For all the RT-PCR analyses, the positive controls (submandibular gland cDNA) yielded products of the predicted size (lane 3 [+] in Figs. 1–3), and the no-template controls were negative, indicating that reaction conditions were optimized and that reagent components were not contaminated (lane 2 [-] in Figs. 1–3). DNA sequencing of representative PCR products confirmed the specific amplification of Klk3, Klk5, and Klk9 with each primer pair. Southern hybridization analysis (Fig. 4) and DNA sequencing were combined to conclusively identify the products of amplification as Klk1 and Klk21. The cDNA dot-blot controls (data not shown) monitored the specificity of these hybridizations. Hybridization of the oligonucleotide probe 21P to the Klk21 cDNA dot was specific with no cross-hybridization to the other kallikreins. The 1P probe for Klk1 hybridized most strongly to the Klk1 cDNA spot, not at all to Klk3 and Klk9, and weakly to Klk5 cDNA. Surprisingly, given the number of differences between the 1P probe and Klk21 sequence (Table 2), this probe also hybridized weakly to the Klk21 cDNA dot. However, since Klk5 was not expressed in these tissues and Klk21 was weakly expressed, this was not considered a problem. Moreover, DNA sequencing of representative PCR products also confirmed the specific amplification of Klk1 and Klk21.
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Embryo Expression
Expression of Klk5 did not occur in the embryo throughout the period studied (Fig. 1d). However, Klk1, Klk3, Klk9, and Klk21 were expressed, with distinct patterns of expression observed (Fig. 1, b, c, e, and f). Expression of Klk3 was detected very weakly at the 2-cell stage. It was not detected at any later stages studied except in one Day 9.5 embryo sample.
Expression of Klk1 was strong in the fertilized egg and 2-cell stage, as detected by Southern hybridization after a short (3 min) exposure to x-ray film (Fig. 1b and Fig. 4a). By the blastocyst stage, expression was essentially undetectable by both RT-PCR and Southern hybridization. Expression of Klk1 was detected again in the Day 9.5 embryos and also in one of two embryos assessed at Day 11, the last day studied.
Like Klk1, Klk9 was expressed in both fertilized egg and 2-cell stages (Fig. 1e). There was no expression in blastocysts, but Klk9 expression was seen in the Day 7.5 embryo, earlier than that seen with Klk1, and was sustained until Day 11.
Expression of Klk21 occurred in all stages between the fertilized egg and Day 7.5 embryo (Fig. 1f) but at very low levels. However, after a lengthy exposure time (2 h), the signal of the Southern hybridization of these RT-PCR products showed a clear pattern of expression. Expression was highest in the 2-cell embryo and then gradually decreased until transcripts were essentially undetected in the embryo at Days 9.5 and 11 (Fig. 4b).
Uterus and Decidua Expression
As with the embryo, Klk5 was not expressed in either the uterus or decidua except in a single Day 1 pregnant uterus sample (Figs. 2d and 3d). Similarly, Klk3 (Figs. 2c and 3c) and Klk9 (Figs. 2e and 3e) were not expressed in the uterus or decidua in a discernible pattern; however, there was very weak expression that was detected in individual samples but was not consistently present in all samples of the same tissue stage.
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There was no expression of Klk1 in the uterus prior to implantation (Day 1 to Day 4). This is best illustrated by the Southern blot (Fig. 4c) of the gel picture in Figure 2b. Although amplification of a product from individual samples at Day 1 and Day 4 of pregnancy stained positive with ethidium bromide (Fig. 2b), on Southern hybridization these products did not hybridize to the probe. The first expression of Klk1 was seen at Day 7.5 of pregnancy in the uterus, consistently present in all samples as detected by both the RT-PCR and Southern hybridization. Although the level of Klk1 expression in samples at the later stages studied was quite varied, expression was generally at lower levels than at Day 7.5. Expression of Klk1 in the decidua (see Figs. 3b and 4e) generally complemented the expression seen in the uterus.
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The temporal expression of Klk21 in the uterus was similar to that of Klk1. At Day 7.5 to Day 11 of pregnancy, Klk21 was consistently expressed in all uterus samples (Fig. 2f and Fig. 4d). The decidua also showed consistent expression of Klk21 that was detected after a shorter exposure time to x-ray film than required for the uterus Southern hybridization (Fig. 4f).
An interesting finding was an additional band that resulted from the amplification of Klk21. This band can be seen most clearly in the positive control of Figure 2f. It was amplified predominantly in uterus samples where Klk21 was present as seen in Figure 2f, on the agarose gel slightly above the identified Klk21 product. DNA sequencing revealed this unknown product to be identical to the Klk21 sequence except for a small insertion and seven single base pair changes (Fig. 5). A BlastA search of all sequences in the GenBank database showed closest sequence homology with Klk21 as expected, but no exact matches. The insertion was positioned at the exon 2-exon 3 junction. The sequence CCGTGAAGA matched the last eight base pairs of the 3' end of intron B, between exons 2 and 3. When this Klk21-like mRNA was translated, the predicted protein stayed in the same reading frame, analogous to the other previously sequenced kallikrein genes, due to the length and position of the insertion, but with an extra three amino acids (Asp-Thr-Ser) (Fig. 5). Five of the seven single nucleotide changes also led to amino acid changes (Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The results show that Klk5 was not expressed in the embryo, uterus, or decidua during the time of study. This is in contrast to findings by Digby [29] of Klk5 expression in the uterus. However, these differences may be attributed to the difference in gene expression between the nonpregnant uterus [29] and pregnant uterus (this study). Alternatively, a lack of specificity of the method employed in the previous study may have resulted in the erroneous identification of Klk5 expression. This is likely, as it is not clear that cross-hybridization was ruled out by methods such as DNA sequencing or dot-blot controls as employed in the present study. It therefore could be worthwhile in future efforts to extend this study to characterize the expression of kallikreins in nonpregnant mice with the current strategy.
All four other kallikreins studied were expressed by the embryo during early development. To date, this is the only characterization of kallikrein expression in the periimplantation embryo. Transcripts for Klk1, Klk3, and Klk9 were detected in the early conceptus (fertilized egg and 2-cell embryos) but not in blastocysts. This suggests that preimplantation expression of these genes is limited to expression from the maternal genome (mRNAs found in oocytes and embryos up to the 2-cell stage are from the maternal genome, as activation of the embryonic genome in the mouse does not occur until the 2-cell stage). In contrast, expression of Klk21 in the early conceptus is continued until Day 7.5 of pregnancy. The expression of genes by the early conceptus suggests involvement in fertilization (for example in sperm-egg interaction) or oocyte maturation. Little is known about the roles of kallikreins in fertilization events. However, Klk9, encoding an EGF-BP, may be involved via its ability to activate EGF, as several studies have shown EGF to accelerate preimplantation development [34] and development of the 2-cell stage to blastocyst [35]. In support of this, EGF is expressed in the mouse uterus early on Day 1 of pregnancy [36].
Expression of Klk9 reappears in the Day 7.5 embryo and is expressed until Day 11, the last day studied. During this time, the embryo is at its peak of invasive activity via expression of many other proteinases, growth factors, and cytokines [1, 2]. EGF has been shown to be necessary to the implanting embryo [36], and it increases secretion of uPA from stromal cells, which is suggested to facilitate the tissue remodeling that occurs with decidualization (see later) [37, 38]. The early embryo does not express its own EGF and is dependent on EGF expressed from the uterus [7]. However, in addition to the EGF receptor [39], we have now shown that the embryo also expresses the EGF-activating protease, EGF-BP (encoded by Klk9), during the most invasive period of the embryo. These results may relate to findings demonstrating that EGF stimulates MMP and uPA secretion from blastocyst outgrowths in vitro [4]. Furthermore, like other kallikreins, EGF-BP has shown ability to act on a range of substrates other than EGF. Uddin and Beg [40] demonstrated that most mouse kallikreins, including mK9 (the protein encoded by Klk9), were able to cleave a specific bond within the bradykinin sequence. EGF-BP has also been shown to activate uPA, an important protease in implantation, as well as macrophage-stimulating protein, which is up-regulated in inflammation [20, 41]. This suggests the possibility that mK9 (also known as EGF-BP) acts in a range of capacities during early development.
Expression of Klk1 reappears later, with strong expression in the Day 9.5 embryo. This time corresponds to the end of invasion and the beginning of organogenesis. The kallikrein-kinin system has been shown to play an important role in the developing kidney by mediating renal growth and differentiation [42]. Kallikrein-generated kinins may play a similar role in the development of other organs. In addition to its primary function of generating the multifunctional peptide bradykinin, tissue kallikrein has been shown to act on a wide range of substrates [15]. MMP requires posttranslational activation, and tissue kallikrein is a protease that has shown ability to activate pro-MMP [17, 18]. Degradation of the ECM in embryo invasion has been largely attributed to MMP expressed by the trophoblast.
Embryonic expression of Klk21 continues to the Day 7.5 embryo, after which it is not expressed again. Although the functional protein encoded by Klk21 has not yet been characterized, the temporal distribution of Klk21 expression in the embryo is consistent with a possible role in the events of early development.
Uterine expression of the mouse kallikreins revealed that these results agree with studies in the rat. Corthorn and Valdes [12] have demonstrated a rise in immunoreactive kallikrein during embryo implantation. Consistent with these findings, our data show a rise in Klk1 (tissue kallikrein) mRNA levels during implantation; Klk1 was expressed in the uterus at highest levels at Day 7.5 of pregnancy, coinciding with the height of trophoblast invasion into the uterus. The embryo commences implantation in response to maternal cues, and its invasive activity is tightly controlled by the uterus, which both promotes and inhibits proteinase expression, stimulating embryonic proliferation but also the transformation of the invasive trophoblast to noninvasive trophoblast giant cells [1, 2]. The expression of Klk1 from the uterus at this time supports the notion that tissue kallikrein may have a role in regulating trophoblast invasiveness via the decidual reaction. The formation of the decidua is characterized by the stimulation of the uterine stromal cells to proliferate and differentiate, an increase in vascular proliferation to such an extent in rodents that implantation nodes are formed, each representing a site of embryo implantation [43]. Macrophages and neutrophils are also attracted to the implantation site [8]. For these reasons, the decidual reaction has been likened to an inflammation reaction, in which the involvement of the kallikrein-kinin system is well acknowledged in many other tissues [15, 16, 44, 45]. The bradykinin B2 receptor, through which bradykinin (the peptide activated by tissue kallikrein) acts, is present in the uterine epithelium and decidual cells [46, 47]. Bradykinin is known to increase capillary permeability and stimulate decidual cells to release cytokines; recently, it has also been shown to have mitogenic effects on decidual cells [48, 49].
The pattern of expression of Klk21 was similar to that of Klk1. However, the decidual expression of Klk21 was notable in that the Southern hybridization signal detecting expression was much brighter than in the uterus with the same time of exposure to x-ray film. The apparent differential expression of the same gene in two closely related but distinct tissue types reflects the characteristic tissue-specific expression of this gene family. It has been proposed that the decidua, as described above, plays a significant role in the control of invasion. As shown by Graham and Lala [1], human decidual cells produce molecules that inhibit trophoblast invasion. Furthermore, Harvey et al. [4] have demonstrated the expression of MMP inhibitors in the decidua immediately adjacent to the invading trophectoderm. The temporal and spatial expression of Klk21 may suggest a role in the control of trophoblast invasion.
Of interest, we have characterized the expression of a Klk21-like mRNA that is expressed in the uterus, but not in the embryo. Single nucleotide changes that lead to amino acid changes support the hypothesis that this is a new gene. The similarity between this novel transcript and Klk21 is consistent with the high nucleotide sequence homology already known to exist within the mouse kallikrein gene family (up to 93%) [31]. This new transcript, in addition to Klk21, may be another mouse kallikrein for which the functional protein has not yet been characterized. It may be relevant to note that in the smaller human kallikrein family, members have been described with functions such as the activation of MMPs and uPA and degradation of IGF-BP3 and the ECM. At present, these functions have not been ascribed to any of the members of the large mouse kallikrein gene family [15].
This is the first report of specific kallikrein expression during early development. The results presented in this study, in conjunction with the well-characterized roles of kallikreins in the regulation of ECM proteinases and growth factors and involvement in many biological functions, strongly implicate the members of the kallikrein gene family in early development. Future studies are aimed at elucidating the specific function and regulation of these genes to enhance our understanding of normal developmental events.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Judith A. Clements, Centre for Molecular Biotechnology, School of Life Sciences, Queensland University of Technology, Gardens Point Campus, 2 George Street, GPO Box 2434, Brisbane, Qld 4001, Australia. FAX: 61 7 3864 1534; j.clements{at}qut.edu.au
Accepted: April 13, 1999.
Received: February 4, 1999.
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