Molecular Human Reproduction, Vol. 5, No. 6, 520-526,
June 1999
© 1999 European Society of Human Reproduction and Embryology
Calpain–calpastatin: a novel, complete calcium-dependent protease system in human spermatozoa
1 Department of Research, Worldwide Medical Corporation, Irvine, California 92618, 2 College of Medicine, University of California Irvine, Irvine, CA 92612, and 3 Department of Basic Sciences, Hitachi Chemical Research Center, Irvine, CA 92612, USA
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Abstract |
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Calpain, a calcium (Ca2+)-activated cysteine protease presents in several somatic mammalian cells, has been demonstrated to mediate specific Ca2+-dependent reactions including cell fusion. Because spermatozoa cells have an absolute Ca2+ requirement for penetration of oocytes, we have postulated that calpain would also be found in mammalian spermatozoa. Here we show that whole sperm homogenate and cell fractions prepared from ejaculated human spermatozoa contain calpain activity. Specific calpain inhibitors impaired this proteolytic activity. Unlike the enzyme described in somatic cells, sperm calpain was mostly particulate in nature and its activity was maximal at pH 9.0. Presence of sperm calpain was confirmed by immunoblot analysis using specific anti-calpain I and anti-calpain II antibodies. A 67 kDa calpain II protein and a 75 kDa calpain I protein were detected. Also spermatozoa contain the endogenous calpain inhibitor, calpastatin. We detected 158.8 ± 24.5 (mean ± SD) fmol calpastatin/mg sperm protein. Immunoblot analysis using specific antibodies showed a 68 kDa calpastatin protein located in the cytosolic fraction. This is the first demonstration that a complete calpain–calpastatin system exists in mammalian spermatozoa. Because calpain is a unique effector system for calcium-dependent processes, our data reveals a novel mechanism by which calcium exerts its regulatory functions in spermatozoa.
calpain/calpastatin/spermatozoa/calcium-dependent proteases/calcium regulation
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Research on fertilization in humans and other mammalian species has revealed that calcium is a key player in the biochemical events that enable spermatozoa to penetrate the oocyte (Yanagimachi, 1988
). One of these events is the membrane fusion process that takes place between the plasma membranes of the sperm cell and the oocyte. Current knowledge indicates that calcium is a signal transducer that specifically mediates membrane fusion following sperm–egg interaction (Yanagimachi, 1988). Accordingly, it has been demonstrated that capacitated spermatozoa in the absence of Ca2+ can collide with and attach to the egg plasma membranes, but are unable to fuse with them (Yanagimachi, 1978, 1982; Wassarmann, 1987). Although the absolute requirement for calcium in this membrane fusion process is well established, our understanding of the mechanisms by which calcium exerts its actions is still limited.
For several years, interesting information about the role played by a calcium-specific protease, called calpain, in the functional activity of membranes has been gathered from a variety of somatic mammalian cells. Calpain is an intracellular, non-lysosomal protease that is isolated from the cytosolic fraction of tissues or cells (Murachi et al., 1981; Croall and Demartino, 1991). The catalytic site contains a cysteine residue which places calpain in the cysteine protease family. Its properties include an absolute dependence on Ca2+ for activity and an optimum pH of 7–8 (Melloni and Pontremoli, 1989; Murachi, 1989). In most mammalian tissues and cells, there are two forms of calpain with different sensitivities to Ca2+, namely, calpain I and calpain II, which require low and high Ca2+ concentrations respectively. The two forms appear to have identical substrate specificity (Murachi, 1989; Croall and Demartino, 1991). Also the two calpains are inhibited by the naturally occurring protein inhibitor, calpastatin (Takano et al., 1988). Calpain figures prominently in the intracellular regulatory activities mediated by calcium, including the calcium-mediated regulation of membrane fusion in somatic cells. Several investigations reveal that some degree of proteolysis must occur for membrane fusion to occur and that a correlation exists between increased membrane fusibility and increased calcium-dependent proteolysis of membrane proteins (Kosower et al., 1983; Schollmeyer, 1986a; Johnson, 1990; Croall and Demartino, 1991). Indeed, there is evidence that calpain degrades membrane proteins and acts by limited proteolysis coupled to transient Ca2+ mobilization (Glaser and Kosower, 1986; Stegmann et al., 1989). Because mammalian spermatozoa have an absolute calcium requirement for certain specific functions such as sperm–egg interaction, we have postulated that a calcium-activated protease functions in spermatozoa. According to our hypothesis, sperm calpain is associated with the cell fusion process that takes place during penetration of the oocyte. However, the existence of a functional calcium-dependent protease, such as calpain, has not been shown in mammalian spermatozoa.
Here, we investigated the presence of calpain in human spermatozoa. For this purpose, we examined cytosolic and sperm particulate fractions and performed enzymatic and immunoblot analysis. In addition, we investigated whether human spermatozoa also contain calpastatin, the endogenous inhibitor of calpain. The results indicate that a calpain–calpastatin system, similar to that described in somatic cells, exists in the sperm cell.
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Materials and methods |
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Sperm preparation
Freshly ejaculated samples of human semen were obtained by masturbation from fertile, healthy men who had normal semen analysis on several occasions. Semen samples contained <5% of cells which were not spermatozoa. Each semen sample was allowed to liquefy and then was centrifuged at 400 g for 10 min, according to methods described previously (Rojas and Bruzzone, 1992
; Rojas et al., 1993). The supernatant was discarded and the sperm cells were washed twice at 200 g for 5 min with 2 ml Ham's F10 medium. The washed spermatozoa were resuspended in a buffer containing 50 mM Tris–HCl pH 7.4, 0.1 mM EGTA and 5 mM 2-mercaptoethanol and homogenized by nitrogen cavitation (1500 psi for 15 min). The homogenate was then centrifuged at 100 000 g for 60 min and the supernatant and pellet were recovered to obtain the cytosol and particulate fraction respectively. The particulate fraction was resuspended in homogenization buffer and the protein concentration of cytosol and particulate fractions were determined by the bicinchonic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA) (Rojas et al., 1992).
Determination of calpain activity
Calpain activity was assayed by a modification of the method of Twining using resorufin-labelled casein (Boehringer Mannheim, Indianapolis, IN, USA) as the substrate (Twining 1984). Unless otherwise indicated, each tube contained sperm homogenate, 50 µl reaction buffer (200 mM Tris, pH 8.5), 10 µl 100 mM CaCl2, 5 mM 2-mercaptoethanol, 25–40 µl 0.4% resofurin-labelled casein in water, and water to make 200 µl final volume. The final concentration of Tris was 50 mM, and Ca2+ 5 mM. The reaction was initiated by the addition of the substrate and incubated for 1 h at 30°C with shaking. At the end of the incubation period, 480 µl of 5% trichloroacetic acid (TCA) was added to each tube. The tube was incubated for an additional 10 min at 30°C. The TCA-precipitated proteins were pelleted at 110 g for 10 min, and 400 µl of the supernatant were transferred to new tubes containing 600 µl of assay buffer (50 mM Tris, pH 8.8). After mixing for 2 min, the fluorescence of the assay mixtures was measured on a fluorometer. Fluorescence values were determined with an excitation filter of 530 nm. Fluorescence values for all samples was obtained the presence and absence of 5 mM CaCl2. Calpain activity was defined as the difference between the two readings expressed as fluorescence units. Also, fluorescence units per mg sperm protein was used when necessary. Each experiment was run in triplicate and was repeated at least three times.
To study the effects of specific inhibitors of calpain activity, several cysteine protease inhibitors with high specificity for calpain were tested including calpain inhibitor I and calpain inhibitor II (Boehringer Mannheim, Indianapolis, IN, USA), and E-64c and EST, also called E64d (Peptide Institute, Osaka, Japan).
Calpain I and calpain II immunoblot analysis
Human sperm cytosol and particulate fraction proteins (50 µg each) and 35 µg each of rat brain cytosol and particulate fraction proteins used as control, were fractionated by sodium dodecyl sulphate –polyacrylamide gel electrophoresis (SDS–PAGE) on 10% gels using the Bio-Rad Mini Protean II electrophoresis system. Gels were run for 1 h and 15 min at 15 mA per gel. Gels were mounted onto nitrocellulose paper membrane (Immobilon NC) and electrotransferred overnight at 50 V in 25 mM Tris, 192 mM glycine, 20% methanol transfer buffer. At the completion of electrotransfer, the nitrocellulose membranes were blocked for 1 h in 0.5% bovine serum albumin in Tris-buffered saline with Tween, (TBST; 50 mM Tris, pH 7.6, 150 mM NaCl, 0.05% Tween 20).
Monoclonal antibodies to human calpain I and polyclonal antibodies to human calpain II were generously provided by Dr John S.Elce, Department of Biochemistry, Queen's University, Kingston, Canada. These antibodies have been fully characterized by Dr Elce's laboratory (Elce et al., 1989; Samis et al., 1991). They have been also used by other investigators for immunocytological localization of calpains and Western blots in somatic cells (Oshima et al., 1989). Each experiment included a sample with non-immune rabbit serum or ascite fluid as controls. Dilutions of 1:1000 and 1:25 000 were usually used for anticalpain I and anticalpain II antibodies respectively. The blots were exposed to the diluted antibodies overnight at 4°C with constant shaking. Calpain I blots were washed three times for 10 min, twice for 5 min in TBST, followed by exposure to 1:100 000 dilution of anti-mouse immunoglobulin (Ig)G conjugated with horseradish peroxidase diluted in TBST for 2 h at room temperature with agitation. Calpain II blots were washed as above, then treated with 1:100 000 anti-rabbit IgG conjugated with horseradish peroxidase. Blots were washed in TBST 3x5 min, 3x10 min, followed by 1x5 min in Tris-buffered saline (TBS; no Tween 20). Blots were developed with the Amersham enhanced chemiluminescence (ECL) detection kit for 1 min. Kodak XAR-5 X-ray film was applied for exposures ranging between 15 s and 5 min. Each experiment was repeated at least five times.
Calpastatin immunoblot analysis
Calpastatin immunoblots were performed essentially as described above, using the same amount of protein loaded onto 10% polyacrylamide gels. The anti-calpastatin antibody used was a mixture of two monoclonal antibodies to human calpastatin obtained from Pierce (Rockford, IL, USA). In combination, these recognize both domain I and II of the calpastatin molecule. The antibodies were diluted 1:16 000 and exposed to the blot overnight at 4°C. The second antibody was sheep anti-mouse IgG–horseradish peroxidase diluted 1:100 000 in TBST. Films were applied for anywhere between 15 s to 15 min. Molecular weight markers were run with every gel. Each experiment was repeated at least five times.
Calpastatin assay
The total calpastatin enzyme immunoassay kit (Pierce, Rockford, IL, USA) was used for determination of calpastatin concentrations in sperm cells. This kit determines the quantity of antigens of total calpastatin by recognizing different epitopes of human calpastatin using monoclonal antibodies. The calpastatin standard provided by the kit is highly purified domain 1 of human calpastatin, which was produced by genetic recombinant techniques. Each experiment was repeated at least three times.
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Results |
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Sperm calpain activity
Effects of sperm protein concentration
Preliminary studies indicated that a significant calpain activity is detected in whole homogenates from ejaculated human spermatozoa. Before detailed studies were undertaken, the rate of enzyme activity was determined at different concentrations of sperm protein. Figure 1
shows that calpain activity was directly proportional to sperm protein up to at least 60 µg protein per assay tube.
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Effects of calcium concentrations and time course studies
The protease activity was highly responsive to changes in calcium concentrations. The effects of Ca2+ on human sperm calpain are illustrated in Figure 2
. Varying concentrations over the range 1–9 mM were added to the assay in the presence of 0.1 mM EGTA. Enzyme activity was progressively enhanced by Ca2+ concentrations up to 5 mM. Higher Ca2+ concentrations, however, resulted in a sharp decline in activity. Similar findings have been reported in somatic cells (Murachi et al., 1981).
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The rate of enzyme activity as a function of time is shown in Figure 3
. Calpain activity in the presence of 50 µg protein homogenate and 3 mM calcium showed a proportional increase with time up to at least 60 min at 30°C.
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Effects of calpain inhibitors
Experiments were then conducted to examine the effects of specific inhibitors of calpain activity. Cysteine protease inhibitors with high specificity for calpain have been developed (McGowan et al, 1989
; Murachi, 1989; Wang, 1990). Preliminary studies indicated that several of these inhibitors used at 0.5 and 1 mM significantly reduced sperm calpain activity. They included calpain inhibitor I, calpain inhibitor II, E-64c and EST (data not shown). Figure 4 illustrates the inhibitory effect of the inhibitor E-64c [trans-epoxysuccinyl-1-leucocylamido (4-guanidino) butano] at a wide range (5–500 µM) of concentrations. A dose-dependent inhibition was observed with increasing concentrations of E-64c. The original calcium protease activity was reduced by 50% (IC50) at 50 µM of the inhibitor.
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Effects of pH
Since calpains in somatic cells show optimal activity at neutral pH (Croall and Demartino, 1991
), we verified this property for sperm calpain. Figure 5 shows the sperm calpain activity as a function of pH. Calpain activity from rat brain was used for control. At low pH, brain and sperm enzymes showed similar properties. Thus, at pH of <6, brain calpain was very low and sperm activity was completely abolished. However, at higher pH there was a marked difference between the two enzymes. Brain calpain showed maximum activity at pH 7.5–8.0 and a decrease at pH >8.0, a profile that is typical of somatic calpain. In contrast, sperm calpain showed progressively higher activity with increasing pH. Activity was maximal at pH 9.0 and remains high at pH 9.5 (Figure 5).
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Cytosolic versus particulate fractions
To examine the subcellular distribution of sperm calpain, we measured its activity in cytosolic and particulate fractions of human spermatozoa. Calpain activity in somatic cells is localized in the cytosol (Murachi, 1989
; Croall and Demartino, 1991). Table I shows that in great contrast to somatic cells, calpain activity in the human spermatozoa is localized mainly in the particulate fraction. Enzyme activity in the cytosolic fraction represented <10% of the sperm activity detected in the particulate fraction.
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Sperm calpain protein
Confirmation of calpain in human spermatozoa was carried out by immunoblot analysis. In these experiments, we used specific antibodies against the two kinds of isozymes described in somatic cells, namely, calpain I and calpain II. Because in our previous experiments on enzyme activity we found a unique subcellular distribution of calpain, we also examined the presence of sperm calpain in particulate and cytosol fractions. Our data indicates that both calpain I and calpain II are present in the human spermatozoa. Accordingly, a 67 kDa calpain II protein described in somatic cells was detected in the sperm cytosol (Figure 6
). As expected for somatic cells, the preparation of rat brain used as control show this protein in the cytosol only (Figure 6). In addition, we found a 75 kDa calpain I protein described in somatic cells in the cytosol and particulate fractions of spermatozoa (Figure 7). These observations agreed with our data on calpain activity.
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Sperm calpastatin
Calpastatin concentrations in human spermatozoa and seminal fluid
We examined the presence of the physiological inhibitor of calpain, calpastatin, to investigate whether a complete calpain–calpastatin system exists in the human sperm cell. Quantitative determinations using a specific immunoassay were performed in 17 human sperm samples. Our data indicate that calpastatin is present in human spermatozoa and that its concentration varies widely (32–378 fmol per mg sperm protein). The mean ± SD value was 158 ± 24 fmol/ mg sperm protein.
To gain information on the origin of sperm calpastatin, we investigated the presence of calpastatin in human seminal fluid. This was pertinent as cysteine protease inhibitors may be present in semen. Initial studies using frozen semen indicated the presence of calpastatin in seminal fluid at a concentrations of 7.8–380.2 fmol per mg seminal fluid protein (data not shown). This calpastatin, however, may derive from sperm leakage due to the freezing–thawing process. Therefore, we also measured calpastatin in seminal fluid from 10 fresh semen samples and from four vasectomized patients. No significant calpastatin was detected in any of these samples, indicating that the levels found in frozen samples were due to sperm leakage. Results are summarized in Table II.
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Immunoblot analysis
We identified sperm calpastatin on the basis of its molecular weight and reactivity with specific antibodies. Also, we determined its subcellular localization. Immunoblot analysis shows that calpastatin is present only in the sperm cytosolic fraction (Figure 8
). The 68 kDa molecular weight band is in agreement with that described in somatic cells (Takano et al., 1988).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In the present study, we have demonstrated the presence of calpain in ejaculated human spermatozoa obtained from normal, fertile men. We characterized its properties and optimized the experimental conditions to quantify the enzyme activities. No calcium-specific protease activity has been reported in mammalian spermatozoa. The proteolytic enzyme acrosin found in membranes of sperm acrosome and considered to be involved in the fertilization process is not responsive to calcium fluxes (Polakoski, 1974
; Schollmeyer, 1986b).
As for calpain described in somatic cells, we found that sperm calpain is dependent on calcium for activation and that its proteolytic activity is effectively reduced by specific calpain inhibitors. Sperm calpain was also confirmed by immunoblot analysis using specific anticalpain antibodies. We found that the human spermatozoa contain both isozymes described in somatic cells, i.e. calpain I and calpain II. These isozymes are considered to represent two forms of calpain with different calcium requirements for activation (Murachi, 1989; Croall and Demartino, 1991). The only data available on the presence of calpain in sperm cells refers to a study in porcine spermatozoa (Schollmeyer 1986b). This investigator identified calpain II in the apical segment of the acrosomal cap as determined by indirect immunofluorescence. Although the report did not demonstrate enzyme activity or a functional calpain protein, detection of this protein is compatible with the observations presented herein.
We have also demonstrated the presence of calpastatin, the endogenous inhibitor of calpain, in human spermatozoa. We detected calpastatin protein by immunoblot analysis and measured its intracellular content. These data indicate that a complete calpain system, i.e. calpain and calpastatin, similar to that described in somatic cells may operate in the sperm cell. The finding is particularly relevant to calpain-mediated actions in membrane fusion. Studies in somatic cell indicate that a limited proteolysis must occur prior to the fusion process (Glaser and Kosower, 1986; Stegmann et al., 1989; Hayashi et al., 1992). The nature of calpain-catalysed proteolysis is thus not digestive, but it proceeds in a controlled manner and results in alteration, rather than simple destruction, of the substrate proteins (Susuki et al., 1987; Johnson, 1990; Hayashi et al., 1992). Therefore, the presence of the natural inhibitor calpastatin provides a physiological mechanism by which a limited calpain proteolysis can occur in sperm cell.
Evidence of calpain in spermatozoa suggests that calcium may act as a second messenger via activation of a specific proteolytic enzyme. This reveals a new mode of calcium action in the sperm cell. The concept is consistent with data from somatic cells indicating that the protease calpain is an effector system for calcium-dependent processes such as membrane fusion (Glaser and Kosower, 1986; Schollmeyer, 1986a; Hayashi et al., 1992). Also because Ca2+ exerts several and highly specialized functions in spermatozoa, this calpain effector system offers new insights on the general mechanisms responsible for calcium action in sperm cells.
Our data suggest that calpain of the human spermatozoa is unique and different from the enzyme described in somatic cells. Accordingly, calpain from a variety of somatic cells show similar characteristics including localization in the cytosol and a neutral pH optimum (pH 7.5). In contrast, we found that sperm calpain is mostly particulate, and its maximal enzyme activity is attained at pH 9.0–9.5. The meaning of the particulate nature of sperm calpain rather than its soluble form is not clear. We found that calpain II protein is present in the cytosol and calpain I protein is present both in the cytosol and particulate fractions. However, we detected most calpain activity in the particulate fraction. It is likely that the reduced enzyme activity in the cytosol is due to inhibition by calpastatin as the endogenous inhibitor, calpastatin, is present only in the soluble fraction. Also, it remains to be clarified whether the subcellular distribution of calpain proteins depends on enzyme activation and calpastatin binding as suggested for somatic calpain (Melloni and Pontremoli, 1989; Murachi, 1989).
On the other hand, the observation that calpain activity is maximal at a higher pH may have physiological relevance as low pH is usually associated with sperm quiescence, while pH>7.5 is associated with sperm stimulation including metabolism and motility (Babcock et al., 1983). It is thus possible that sperm calpain activity becomes maximally expressed at the time the sperm cell is fully active. This would agree with the key role that the sperm calpain may play during sperm–egg interaction.
Demonstration of a complete calpain–calpastatin system in human spermatozoa supports our hypothesis that the protease may participate in a calcium-dependent process such as sperm–egg fusion. Based on data in somatic cells, we speculate that the influx of Ca2+ associated with the fertilization process induces a calpain–dependent proteolysis which promotes fusibility in the sperm membrane. Removal or alteration of specific fusion proteins will produce a lipid-rich area which makes sperm and egg membranes competent for fusion. In this model, we conceive calpastatin as being involved in preventing calpain activation prior to fertilization and in regulating calpain proteolysis during the fusion events. Additional support for this hypothesis may derive from studies designed to identify the specific relevant substrates of sperm calpain and to demonstrate that inhibition of the enzyme has detrimental effects upon the capacity of the sperm cell to penetrate the egg.
Because fusion and penetration of the egg by the sperm cell is absolutely dependent on calcium, it is reasonable to assume that membrane fusion is a primary target for sperm calpain action. However, we cannot disregard the possibility that the enzyme also participates in other calcium-mediated actions besides sperm–egg fusion. Studies in somatic cells suggest that calpain modulates intracellular calcium homeostasis in a variety of cells and is selective for a subset of cellular proteins including cytoskeletal proteins, membrane receptors, and enzymes, such as protein kinase C, which participates in signal transduction (Melloni and Pontremoli, 1989; Murachi, 1989; Croall and Demartino, 1991). Thus, it is feasible that the enzyme may be involved in functions such as sperm capacitation and acrosome reaction which are also dependent on mobilization of cellular Ca2+.
In conclusion, we have conducted enzymatic and immunoblot studies and demonstrated for the first time that mammalian spermatozoa contain a complete calpain–calpastatin system. Because calpain has been shown to play a key role in fusibility of membranes in a variety of somatic cells, we envisage sperm calpain as being associated with sperm–egg fusion, a process which is absolutely dependent on calcium. The enzyme system, however, may also participate in other calcium-dependent events in the spermatozoa. Further studies are expected to elucidate these possibilities. Whatever the target of calpain action, the existence of a calpain–calpastatin system reveals a novel mechanism by which calcium exerts its regulatory functions in the human spermatozoa. Exploring this mechanism may lead to new modalities of human contraception and treatment of male infertility.
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Acknowledgments |
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We thank Dr John S.Elce, Queen's University, Kingston, Canada, for generously providing the anticalpain antibodies. This research was supported in part by a World Health Organization grant, WHO project # 93063.
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Notes |
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4 To whom correspondence should be addresed at WMED, 199 Technology Drive, 150 Irvine, CA 92618, USA
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References |
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Submitted on April 6, 1998; accepted on March 8, 1999.
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