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Biology of Reproduction 60, 551-557 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Canine Preprorelaxin: Nucleic Acid Sequence and Localization within the Canine Placenta

T. Klonisch1,a, S. Hombach-Klonischa, C. Froehlicha, J. Kauffoldb, K. Stegera, B.G. Steinetzc, and B. Fischera

a Department of Anatomy and Cell Biology, Faculty of Medicine, Martin-Luther-University Halle-Wittenberg, D-06097 Halle (Saale), Germany b Large Animal Clinic for Theriogenology and Ambulance Services, Faculty of Veterinary Medicine, University of Leipzig, Leipzig, Germany c Nelson Institute of Environmental Medicine, New York University Medical Center, Tuxedo, New York 10987


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Employing uteroplacental tissue at Day 35 of gestation, we determined the nucleic acid sequence of canine preprorelaxin using reverse transcription- and rapid amplification of cDNA ends-polymerase chain reaction. Canine preprorelaxin cDNA consisted of 534 base pairs encoding a protein of 177 amino acids with a signal peptide of 25 amino acids (aa), a B domain of 35 aa, a C domain of 93 aa, and an A domain of 24 aa. The putative receptor binding region in the N'-terminal part of the canine relaxin B domain GRDYVR contained two substitutions from the classical motif (E->D and L->Y). Canine preprorelaxin shared highest homology with porcine and equine preprorelaxin. Northern analysis revealed a 1-kilobase transcript present in total RNA of canine uteroplacental tissue but not of kidney tissue. Uteroplacental tissue from two bitches each at Days 30 and 35 of gestation were studied by in situ hybridization to localize relaxin mRNA. Immunohistochemistry for relaxin, cytokeratin, vimentin, and von Willebrand factor was performed on uteroplacental tissue at Day 30 of gestation. The basal cell layer at the core of the chorionic villi was devoid of relaxin mRNA and immunoreactive relaxin or vimentin but was immunopositive for cytokeratin and identified as cytotrophoblast cells. The cell layer surrounding the chorionic villi displayed specific hybridization signals for relaxin mRNA and immunoreactivity for relaxin and cytokeratin but not for vimentin, and was identified as syncytiotrophoblast. Those areas of the chorioallantoic tissue with most intense relaxin immunoreactivity were highly vascularized as demonstrated by immunoreactive von Willebrand factor expressed on vascular endothelium. The uterine glands and nonplacental uterine areas of the canine zonary girdle placenta were devoid of relaxin mRNA and relaxin. We conclude that the syncytiotrophoblast is the source of relaxin in the canine placenta.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The heterodimeric polypeptide relaxin is a member of the insulin-like superfamily and a hormone of pregnancy in numerous species [1]. In the dog, relaxin is undetectable in the serum of cycling, pseudopregnant. or hysterectomized bitches and male dogs [2–4]. In the pregnant bitch, both the placenta and the ovary secrete relaxin, with the placenta being the major contributor to the serum relaxin levels [3, 5]. During the approximately 60 days of gestation, immunoreactive relaxin is first detected in the serum by Day 18 of gestation and exhibits the highest serum relaxin concentrations of any species reported to date, reaching peak levels at 6–8 wk of gestation [2, 4]. Relaxin is regarded as a specific marker of pregnancy in dogs [3, 4, 6]. Upon parturition or shortly before spontaneous abortion, relaxin serum concentrations decrease to undetectable levels, implicating relaxin as a potentially useful indicator for monitoring the onset of parturition in this species [4]. Recently, the amino acid sequence of the A and B domains of canine relaxin have been determined [5]. Employing solid-phase peptide synthesis and sequential site-directed disulfide bond formation, we have produced synthetic bioactive dog relaxin, but the procedure proved to be difficult and inefficient, yielding only low amounts of relaxin [4, 7]. Information is lacking on the nucleic acid sequence of canine relaxin and the cellular source of relaxin at the uteroplacental interface of the endotheliochorial placenta of the dog. Employing uteroplacental tissues at approximately Days 30 and 35 of gestation, we have determined the nucleic acid sequence of canine preprorelaxin and demonstrate the cellular localization of relaxin in the canine placenta.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Collection of Tissues and RNA Isolation

We employed uteroplacental tissues from four pregnant domestic dogs (Canis domesticus) at approximately Days 30 (n = 2) and 35 (n = 2) of gestation as determined by measurement of the crown-rump length of the fetus. All canine tissues used were obtained during the course of medically indicated procedures, and no animal was operated upon or killed specifically for this study. Canine kidney served as control tissue and was collected at a local clinic from a male whelp killed in a road accident. The uteroplacental tissue at Day 30 of gestation was fixed in Bouin's solution and embedded in paraffin. Uteroplacental tissue at Day 35 of gestation and kidney tissue were snap-frozen in liquid nitrogen and used for isolation of total RNA with Trizol reagent (Life Technologies, Eggenstein, Germany). For the cloning of canine preprorelaxin, mRNA was isolated from 75 µg of total uteroplacental RNA using oligo(dT)-coated magnetic beads (Dynal, Hamburg, Germany). The amount of mRNA isolated was determined spectrophotometrically at 260 and 280 nm [8].

Cloning of Canine Preprorelaxin by RT- and RACE-PCR

Reverse transcription (RT)-polymerase chain reaction (PCR) and rapid amplification of 5'- and 3'-cDNA ends (5'- and 3'-RACE)-PCR were performed on mRNA of canine uteroplacental tissue at Day 35 of gestation to determine the cDNA for canine preprorelaxin. All PCR primers employed in this study (Fig. 1; Table 1) flanked the putative single intron at the N terminus of the C domain of relaxin to preclude any genomic DNA amplification (Figs. 1 and 2). Using approximately 600 ng of mRNA, first-strand cDNA synthesis was done using the Superscript reverse transcriptase kit (Life Technologies). PCR reactions were carried out in 50 µl solution containing 1 µl of cDNA, 5 µl of 10-strength Advantage cDNA polymerase mix buffer, 100 µM of dNTP, 10 pmol of each primer, and 2.5 U Advantage cDNA mix polymerase (Clontech, Heidelberg, Germany). For the initial amplification of a cDNA fragment of canine relaxin, we used a degenerate oligonucleotide primer pair designed according to the published amino acid sequence of the A and B domains of canine relaxin (primers 1 and 2; Figs. 1 and 2; Table 1) [5]. The PCR cycles consisted of an initial denaturation for 3 min at 95°C, followed by 40 cycles of denaturation at 95°C and annealing at 56°C, both for 1 min each; an elongation step for 2 min at 72°C; and a final extension cycle for 10 min at 72°C. Employing gene-specific primers (GSP; Table 1) in combination with a universal primer (primer 10; Figs. 1 and 2; Table 1) supplied with the 5'- and 3'-RACE-PCR kits (Life Technologies), the missing cDNA coding sequences to the 5' and 3' ends (primers 6–7 and primers 3–5, respectively; Figs. 1 and 2; Table 1) of canine preprorelaxin were amplified for 35 cycles at an annealing temperature of 68°C. Finally, using RT-PCR for 40 cycles at an annealing temperature of 68°C and a specific primer pair located at both ends of the coding sequence (primers 8 and 9; Figs. 1 and 2; Table 1), the complete cDNA of canine preprorelaxin was amplified. All PCR products were separated on a 1% low-melting point agarose gel, purified by Magic column extraction, and cloned into the pGEM-T vector (both Promega, Heidelberg, Germany). Sequence analysis of the PCR-amplified cDNA clones was performed employing the PRISM dye Deoxy Terminator cycle sequencing kit (Perkin Elmer, Foster City, CA) and T7 or SP6 sequencing primers.



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  		FIG. 1. Nucleic acid sequence and deduced amino acid sequence of canine preprorelaxin cloned from uteroplacental tissue at Day 35 of gestation. The positions of the primers used for RT- and RACE-PCR are indicated. * Putative intron site.


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  		TABLE 1. List of primers employed for RT-PCR and 5'- and 3'-RACE-PCR, and their binding sites in relation to the nucleic acid sequence of canine preprorelaxin.



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  		FIG. 2. Schematic representation of canine preprorelaxin. Numbered arrows indicate the locations of the primers (Table 1) used to clone the cDNA employing RT- and RACE-PCR techniques.

Northern Analysis

Twenty micrograms of total RNA extracted from canine uteroplacental tissue at Day 35 of gestation, and kidney were run on a 1% formaldehyde agarose gel containing ethidium bromide to assess equal loading by comparison of the rRNA bands. RNA molecular weight markers (RNA ladder 0.44–1.77 kilobases [kb]; Life Technologies) were included with the gel. After blotting for 1.5 h by the downward alkaline blotting technique [9], nylon membranes (Hybond N+; Amersham, Braunschweig, Germany) were prehybridized for 30 min at 62°C with Easy-Hyb hybridization buffer (Boehringer Mannheim, Mannheim, Germany) and incubated overnight at 62°C in 15 ml of the same solution containing approximately 200 ng/ml of digoxigenin (DIG)-labeled cRNA probe. Membranes were washed 2 x 20 min in double-strength SSC (single-strength SSC is 0.15 M sodium chloride, 0.015 M sodium citrate)/0.1% SDS at room temperature, followed by 2 x 20 min in 0.5-strength SSC/0.1% SDS at 58°C. Membranes were processed with the DIG luminescence detection kit (Boehringer Mannheim), and specific hybridization signals were visualized by chromogenic membrane staining with nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Sigma, Deisenhofen, Germany).

DIG-Labeling of cRNA and In Situ Hybridization

For cRNA synthesis, 5 µg of the pGEM-T plasmid clone containing the insert for canine prorelaxin were digested with the restriction enzymes NcoI (antisense cRNA) and NotI (sense cRNA; both New England Biolabs; Schwalbach/Taunus; Germany), phenol-extracted, and precipitated. Digested and extracted plasmid at 1 µg was used for cRNA synthesis employing a cRNA synthesis kit (AMS Biotechnology, Wiesbaden, Germany) and a 10-strength DIG-RNA labeling mix (Boehringer Mannheim). After precipitation of the DIG-labeled cRNA, the pellet was dissolved in 70 µl of diethyl pyrocarbonate (DEPC)-water, and the quantity of cRNA was determined spectrophotometrically at 260 and 280 nm and by dot blot analysis of serial dilutions of DIG-labeled cRNA [10]. Before nonradioactive in situ hybridization, canine uteroplacental sections (6 µm thick) were attached to glass slides coated with 2% aminopropyltriethoxysilane (APTEX; Sigma). For in situ hybridization, we employed paraffin-embedded and cryocut uteroplacental sections at Days 30 and 35 of gestation, respectively. Paraffin-embedded sections were dewaxed, digested with 30 µg/ml proteinase K (Boehringer Mannheim), postfixed with 4% paraformaldehyde, and then processed similarly to cryocut sections [11]. A 1:1000 dilution of an anti-DIG alkaline phosphatase-conjugated Fab-antibody (Boehringer Mannheim) in 1% BSA was employed for the detection of DIG-labeled RNA. Specific signals were visualized using the chromogen combination NBT/BCIP. After counterstaining with hematoxylin, the slides were mounted in glycerol gel and examined under brightfield microscopy.

Immunohistochemistry

Immunohistochemical staining was performed on 6-µm-thick paraffin sections of canine uteroplacental tissue at Day 30 of gestation mounted on APTEX-coated slides. Dewaxed and rehydrated sections were incubated with proteinase K (Boehringer Mannheim) at 30 µg/ml for 30 min at 37°C, and endogenous alkaline phosphatase was inactivated with 20% acetic acid. Tissue sections were blocked with 3% BSA in 0.5 M Tris-buffered saline (TBS) for 30 min to saturate nonspecific binding sites. The primary mouse monoclonal antibodies to cytokeratin at 1:250 (MNF 116; Dako, Hamburg, Germany) and vimentin at 1:2000 (V9; Dako) and the primary rabbit polyclonal antibody to von Willenbrand factor at 1:100 (P-0226; Dako) as well as the polyclonal antiserum to dog relaxin (No. 78513) [4] at 1:250 were diluted in 0.5 M TBS containing 3% BSA plus 3% nonimmune goat normal serum (Dianova, Hamburg, Germany). Negative controls were performed in all cases by omitting the primary antibody. After overnight incubation at 4°C, sections were washed 3 x 5 min in 0.1 M TBS and incubated with alkaline phosphatase-conjugated goat anti-mouse at 1:50 or alkaline phosphatase-conjugated goat anti-rabbit at 1:100 (both Dianova), respectively, diluted in 0.5 M TBS containing 1% BSA, for 1 h at room temperature. After being washed 3 x 5 min in 0.1 M TBS, sections were incubated for 30 min with a mouse alkaline phosphatase anti-alkaline phosphatase complex (APAAP; Dako) or rabbit APAAP (Sigma), respectively, both at 1:100 in 0.5 M TBS. Incubations with the secondary antibodies and the APAAP complexes were repeated, and immunostaining was visualized with the Histo Mark Red AP substrate (KPL, Gaithersburg, MD). Sections were counterstained with hematoxylin, mounted with glycerol gel, and examined under brightfield microscopy.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Performing RT-PCR and RACE-PCR on mRNA isolated from uteroplacental tissue at Day 35 of gestation, we cloned the cDNA for canine preprorelaxin, which consisted of 534 basepairs (bp) encoding 177 amino acids (aa). According to the known cleavage sites in other relaxin molecules [1], canine preprorelaxin encoded a signal peptide of 25 aa, a B domain of 35 aa, a C domain of 93 aa, and an A domain of 24 aa, respectively (Figs. 1 and 2). Control experiments using either mRNA or water as templates in the PCR reactions yielded no amplification products (data not shown). The peptide sequence of the A and B domains as deduced from the nucleic acid sequences was identical to the one obtained by amino acid sequence analysis of canine relaxin purified from placental tissues [5]. The locations of the cysteine residues in both the A and B domains were conserved, and the putative receptor binding peptide sequence GRDYVR in the B domain contained two substitutions from the classical motif (E->D and L->Y). The deduced amino acid sequence of canine preprorelaxin shared highest homology with porcine (52.3%) and equine (50.0%) preprorelaxin, respectively (Table 2) [12, 13]. Among the different relaxin domains, the signal peptide was most conserved, displaying an amino acid homology of 72% and 64% with the corresponding signal peptide of porcine and equine preprorelaxin (Table 2). When equal amounts of total RNA from uteroplacental tissue at Day 35 of gestation and dog kidney were processed for Northern analysis, a single specific hybridization signal at 1 kb was exclusively detected in the uteroplacental RNA (Fig. 3). Nonradioactive in situ hybridizations were performed on paraffin-embedded uteroplacental tissue sections at Day 30 and similar cryocut sections at Day 35 of gestation. Of the four pregnant bitches studied, specific hybridization signals for relaxin mRNA were exclusively detected in the zonary placental girdle but were absent in the paraplacental regions (data not shown). Maternal endometrium and all sections treated with the DIG-labeled sense cRNA were also devoid of relaxin mRNA (Fig. 4C). Within the placental girdle, the basal cell layer of the chorionic villi penetrating the maternal endometrium was devoid of relaxin mRNA, immunoreactive relaxin, and vimentin (Fig. 4, A, B, and H) but expressed immunoreactive cytokeratin (Fig. 4G). These cells were identified as cytotrophoblast cells. Immediately surrounding the fetal chorionic villi, an extensive cell layer displayed specific hybridization signals for relaxin mRNA. While cryosections of uteroplacental tissue at Day 35 of gestation showed intense hybridization signals at the fetal aspect of the chorioallantois (data not shown), the paraffin sections of uteroplacental tissue at Day 30 of gestation displayed weaker web-like staining in the same areas but stronger hybridization signals at the tip of the penetrating chorionic villi (Fig. 4, A and B). The differences in the intensity of hybridization signals in cryocut or paraffin-embedded placental sections were most likely due to the fixation method employed, as we always observed a similar general distribution of relaxin mRNA in all four placentae studied. Increasing the proteinase K concentration did not result in stronger hybridization signals in this area of the placental labyrinth. When the rabbit antiserum No. 79813 specific for dog relaxin [4] was employed on uteroplacental tissue at Day 30 of gestation, relaxin immunoreactivity was colocalized in those areas of the chorioallantoic tissue expressing relaxin mRNA (Fig. 4, D and E). Despite weaker hybridization signals for relaxin mRNA at the fetal aspect of the chorioallantois, we observed strong immunoreactivity for relaxin in these placental areas (Fig. 4D). The cells expressing relaxin mRNA and immunoreactive relaxin also immunostained positive for cytokeratin (Fig. 4G) and negative for vimentin (Fig. 4H), and were, therefore, identified as syncytiotrophoblast. Immunostaining against von Willenbrand factor, a marker for vascular endothelial cells, revealed intense vascularization in those areas of the canine placenta rich in immunoreactive relaxin (Fig. 4I; see also Fig. 4D). Control experiments omitting the primary antibodies consistently gave negative results (Fig. 4F).


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  		TABLE 2. Homology of canine preprorelaxin and single relaxin domains with relaxin molecules of other species.



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  		FIG. 3. Northern analysis was performed employing 20 µg of total RNA extracted from dog kidney (lane 1) and uteroplacental tissue at Day 35 of gestation (lane 2). Equal amounts of total RNA were loaded onto a 1% formaldehyde gel as illustrated by ethidium bromide staining of the 18S bands (data not shown). Hybridization was performed at 62°C, and stringent washing was done 2 x 20 min in 0.5-strength SSC at 58°C. Upon colorimetric detection, a specific 1-kb relaxin transcript was visualized in total RNA of uteroplacental tissue (lane 2). No hybridization signal for relaxin transcripts was obtained in total RNA extracted from kidney tissue (lane 1). The Northern blot was scanned for presentation.



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  		FIG. 4. Photomicrographs of paraffin-embedded sections of the feto-maternal interface of a pregnant bitch at Day 30 of gestation subjected to nonradioactive in situ hybridization for the detection of canine relaxin mRNA expression (A–C), and rabbit or mouse APAAP indirect immunohistochemistry with a rabbit polyclonal antibody specific for dog relaxin [14] (D–F), with mouse monoclonal antibodies specific for cytokeratin (G) or vimentin (H), and with a rabbit polyclonal antibody to von-Willebrand factor (I). Specific hybridization signals for relaxin mRNA were observed in the syncytiotrophoblast of the canine placental labyrinth surrounding the chorionic villi (v) (A, B). Maternal endometrium, nonplacental uterine areas, and tissue sections treated with the sense cRNA relaxin probe were devoid of hybridization signals (C). Immunoreactive relaxin was colocalized to the trophoblastic syncytium (D, E), which expressed relaxin mRNA and immunoreactive cytokeratin (G) but not vimentin (H), confirming the trophoblast identity of the cells expressing relaxin. Immunoreactive vimentin was strongly expressed by endometrial stromal cells (not shown); vascular endothelium was weakly stained (arrow, H). Von Willebrand factor was immunolocalized to a dense vascular network at the fetal aspect of the placental labyrinth containing the syncytium highly expressing relaxin (I). The arrow indicates a maternal blood vessel. A typical negative control section (F) lacks the primary antibody. Magnification: A, C, D, F, H, I: x131; B, E, G: x262 (reproduced at 97%).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Relaxin is an early product of the dog conceptus, and of all species studied so far, the pregnant bitch contains the highest serum relaxin levels, reaching up to 10 µg/ml [4]. As a first step towards elucidating the role of relaxin during placentation in the dog, we have determined the nucleic acid sequence of canine relaxin from uteroplacental tissue at Day 35 of gestation. At this stage of pregnancy, the canine placenta is well established, and serum relaxin immunoreactivity reaches peak levels [4]. Amino acid sequence analysis of placentally derived canine relaxin recently revealed an A domain consisting of 24 residues and a B domain ranging in length from 24 to 35 amino acids [5]. This heterogeneity in length of the B domain probably reflected partial C-terminal enzymatic cleavage as we determined a single amplification product of 534 bp that also excluded the presence of alternatively spliced relaxin transcripts recently demonstrated for the human relaxin genes [14]. The N terminus of the B domain contained a putative signal peptide cleavage site at Ala25 [15, 16], and, at the C/A domain junction of canine prorelaxin, we detected the sequence RKKR, which is highly conserved among various prorelaxin molecules and a well-known consensus cleavage motif for furin (Fig. 1) [1, 17, 18]. Consisting of 93 amino acid residues, the C domain of canine relaxin was considerably shorter than the sizes reported for the C domain in relaxin of the guinea pig and mouse (102 residues) [19, 20]; pig, human (H1 and H2), and marmoset monkey (104 residues) [12, 21–23]; rat (105 residues) [24]; equine species (109 residues) [25], and tammar wallaby (111 residues) [26]. The detection of a 1-kb transcript encoding for preprorelaxin in placental tissue was in agreement with earlier reports on the size of relaxin mRNA transcripts in other species [12, 19, 20, 25, 27]. Unlike in mouse [20], horse [13], and human [22] relaxin, no additional transcripts were detected in canine relaxin.

In the pregnant bitch, relaxin serum immunoreactivity becomes detectable shortly after the beginning of implantation at Day 18 of gestation [4], when primary chorionic villi begin to form as pseudopodia-like extensions from the cells of the trophectoderm [28]. Extensive branching of these villi results in multiple secondary villi, which finally create the labyrinthine endotheliochorial dog placenta, known to be the major source for relaxin during the approximately 60 days of gestation in the dog [5, 29]. Immediately after the onset of the endotheliochorial placentation in the dog, a trophoblastic syncytium forms around the chorionic villi [30], which we have identified as the source for relaxin mRNA and relaxin in the canine placenta. The multinucleated syncytiotrophoblast of the rabbit placenta has previously been shown to store and express SQ10, a molecule structurally related to relaxin [31, 32]. In the human placenta, the syncytiotrophoblast of the decidua basalis and the cytotrophoblast of the smooth chorion have been identified as a source for H1 and H2 relaxin [33, 34]. Canine cytotrophoblast cells at the core of the chorionic villi penetrating the maternal endometrium along the lumen and into the mouths of the uterine glands [30] were devoid of relaxin. In the equine placenta, we have previously identified a population of trophoblast cells not expressing relaxin but instead presenting molecules of the major histocompatibility complex (MHC) class I on their cell surfaces [35, 36]. These pseudostratified trophoblast cells differed in morphology from the relaxin-expressing trophoblast cells of the microcotyledons. The canine cytotrophoblast cells devoid of relaxin also displayed a different morphology compared to that of the surrounding syncytiotrophoblast expressing relaxin. Whether the canine cytotrophoblast expresses MHC class I molecules is currently under investigation.

The functional role of relaxin in the placenta of any species is unknown. Low relaxin serum immunoreactivity is associated with complications during pregnancy in various species [4, 37, 38], and in women [39]. Relaxin has growth-promoting potential on rat uterine, cervical, and vaginal tissues [40, 41] and is expressed by highly proliferative fetal tissues in the dog placenta, further supporting our hypothesis that relaxin is a placental growth factor [35]. The expanding placenta requires sufficient blood supply for rapid growth. In canine placentae at midterm, we observed extensive vascularization at the fetal aspect of the placental labyrinth containing the syncytiotrophoblast, which, at this stage of pregnancy, secretes large quantities of relaxin [4]. Reports on a possible angiogenic effect of relaxin are controversial [42, 43]. However, relaxin in synergy with estrogen and progesterone was shown to have vasodilatory properties [44, 45] and affects uteroplacental blood flow during early pregnancy in humans [46]. Further work is needed to elucidate the effects relaxin may have on the uteroplacental vasculature during pregnancy in the bitch.


   ACKNOWLEDGMENTS
 
The authors would like to thank Dr. Wenkel at the Animal Clinic, Martin Luther University Halle-Wittenberg, for excellent cooperation.


   FOOTNOTES
 
1 Correspondence: T. Klonisch, Department of Anatomy and Cell Biology, Martin-Luther-University Halle-Wittenberg, Grosse Steinstr. 52, D-06097 Halle (Saale), Germany. FAX: 0049 345 557 1700; thomas.klonisch{at}medizin.uni-halle.de Back

Accepted: October 2, 1998.

Received: April 27, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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