Quantitative trait locus mapping of genes regulating pulmonary PKC activity and PKC-alpha content

Lori D. Dwyer-Nield1, Beverly Paigen2, Stephanie E. Porter1, and Alvin M. Malkinson1

1 Department of Pharmaceutical Sciences and University of Colorado Cancer Center, University of Colorado Health Sciences Center, Denver, Colorado 80262; and 2 Jackson Laboratory, Bar Harbor, Maine 04609


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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Strain A/J mice, which are predisposed to experimentally induced asthma and adenocarcinoma, have the lowest pulmonary protein kinase (PK) C activity and content among 22 inbred mouse strains. PKC in neonatal A/J mice is similar to that in other strains, so this difference reflects strain-dependent postnatal regulation. PKC activity is 60% higher in C57BL/6J (B6) than in A/J lungs, and the protein and mRNA concentrations of PKC-alpha , the major pulmonary PKC isozyme, are two- to threefold higher in B6 mice. These differences result from more than a single gene as assessed in F1, F2, and backcross progeny of B6 and A/J parents. Quantitative trait locus (QTL) analysis of 23 A×B and B×A recombinant inbred strains derived from B6 and A/J progenitors indicates a major locus regulating lung PKC-alpha content that maps near the Pkcalpha structural gene on chromosome 11 (D11MIT333; likelihood ratio statistic = 12.5) and a major locus controlling PKC activity that maps on chromosome 3 (D3MIT19; likelihood ratio statistic = 15.4). The chromosome 11 QTL responsible for low PKC-alpha content falls within QTLs for susceptibilities to lung tumorigenesis and ozone-induced toxicity.

protein kinase C; inbred mouse strains; developmental regulation; genetic mapping; lung tumor susceptibility


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

STRAIN A/J mice are more susceptible than other strains to pulmonary damage including lung tumorigenesis (21) and ozone-induced lung injury (31). For example, nearly 100% of A/J mice develop one or a few lung adenomas spontaneously by the end of their lifetime and several dozen adenomas within a few months after injection of a carcinogen such as urethan. In contrast, no tumors appear in untreated C57BL/6J (B6) mice, and less than half of the B6 mice injected with urethan develop even a single tumor. Strain-dependent lung tumor susceptibilities display a continuum ranging from the highly sensitive A/J strain to resistant strains such as B6. At least 14 genetic loci, variously designated as Pas (pulmonary adenoma susceptibility), Par (pulmonary adenoma resistance), and Sluc (susceptibility to lung cancer), have been assigned to chromosomal locations (20). The mapping techniques used include recombinant inbred (RI) (29), quantitative trait locus (QTL) (12), and recombinant congenic strains (11). With the exception of the K-ras protooncogene as Pas1 (17), the identities of these genes have not been firmly established. Identifying gene products that confer resistance to lung cancer would provide new tools with the potential for chemoprevention and therapy.

The pulmonary content and activity of protein kinase (PK) C may contribute to these strain differences in susceptibility to lung tumorigenesis. A proposed candidate for Par3, a pulmonary adenoma resistance gene on chromosome 18, is the PKC isozyme PKC-eta (29). PKC regulates pulmonary differentiation and is also associated with proliferative quiescence. PKC activation causes immortalized, nontumorigenic lung epithelial cells to round up (28), a morphological change that is facilitated by altered cytoskeletal architecture (7) and terminated by calpain-mediated proteolysis of PKC-alpha (6). PKC activity increases when these cultured lung cells undergo density-dependent growth inhibition (4), and PKC-alpha content decreases on neoplastic transformation (6). During the compensatory hyperplasia that repairs chemically damaged lungs, both PKC activity (23) and PKC-alpha content (26) decrease.

Thus it is of great interest that A/J lungs have less PKC activity (22) and content (24) than lungs from other inbred strains of mice. These variations reflect a developmental difference because the pulmonary PKC concentration and activity in A/J mice were similar at birth to those in BALB/cBy (CBy) mice, but CBy PKC content and activity increased postnatally to a greater extent than those in A/J mice (24). This difference was observed in isolated Clara cells as well as in whole lung extracts (4). The Clara cells and alveolar type 2 pneumocytes, stem cells of their respective compartments (8, 9), are the probable precursor cells of mouse lung tumors (19). Consistent with this, the basal rates of Clara and type 2 cell proliferation in various strains reflect their relative lung tumor susceptibilities (35).

Herein we describe the following: 1) of 22 inbred strains, only adult A/J mice exhibit both low PKC activity and content in the lung; 2) PKC-alpha is the isozyme responsible for this strain difference in PKC content; 3) PKC-alpha protein variation is a consequence of differential PKC-alpha mRNA concentrations; 4) standard Mendelian crosses between A/J and B6 mice suggest that multiple genes determine this difference; and 5) two loci consisting of a major locus that regulates PKC-alpha content on chromosome 11 near the PKC-alpha structural gene and a locus on chromosome 3 that affects PKC activity were identified by QTL analysis of A×B and B×A RI strains. QTLs for susceptibility to lung tumorigenesis (10) and ozone-induced lung toxicity (31) have been reported at the chromosome 11 Pkcalpha site.


    METHODS
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INTRODUCTION
METHODS
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DISCUSSION
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Materials. A/J, B6, A×B and B×A RI strains, CBy, NZW/J, LP/J, P/J, PL/J, CE/J, SM/J, 129/J, RF/J, MOLD/RKIII, RIII/SmMOB, CAST/Ei, DBA/2J, YBR, SK, CXB 3, CXB 4, and Mus spretus mice were purchased from Jackson Laboratory (Bar Harbor, ME). GRS/N mice were obtained from the National Institutes of Health (Bethesda, MD). SS/N mice were a generous gift from Dr. Susan Fischer (University of Texas, Smithville, TX). AB6F1, AB6F2, (A×B6)F1 × A, and (A×B6)F1 × B6 backcross mice were bred in our facility at the University of Colorado (Denver). Mice were maintained on aspen chip hardwood bedding (Northeastern Products, Warrenburg, NY), fed Wayne Rodent Blox (Teklad Premier, Madison, WI) and tap water ad libitum, and kept on a 12:12-h light-dark cycle. Pulmonary PKC activities are maximal by 6 wk of age (22). Male and female mice 6-14 wk of age were used interchangeably because no gender differences in PKC activity were observed. Pan-PKC antibody was kindly provided by Dr. Curtis Ashendale (Purdue University, West Lafayette, IN). PKC-alpha antibody was purchased from Upstate Biotechnology (Lake Placid, NY), and PKC-beta , PKC-gamma , PKC-delta , PKC-varepsilon , and PKC-zeta antibodies and alkaline phosphatase-conjugated anti-mouse IgG and anti-rabbit IgG secondary antibodies were from Life Technologies (Gaithersburg, MD). Immobilon-P polyvinylidene difluoride membranes were purchased from Millipore (Bedford, MA), and Chemilume 490 was from Lumigen (Detroit, MI). Bovine brain PKC-alpha cDNA (pbPKC-alpha 21), encompassing nucleotides 590-1120 of the published sequence (encodes the calcium and ATP binding sites), was purchased from American Type Culture Collection (Manassas, VA). All other chemicals were purchased from Sigma (St. Louis, MO).

Sample preparation. Lungs were perfused as previously described (25) and solubilized in homogenization buffer [20 mM HEPES (pH 7.5), 2 mM EDTA, 2 mM EGTA, 1 mM dithiothreitol, and 10% (vol/vol) glycerol] containing freshly made protease inhibitors (5 µg/ml of aprotinin, 10 µM leupeptin, and 1 mM phenylmethylsulfonyl fluoride). Soluble (cytosolic) and particulate fractions were separated by a 30-min, 16,000 g centrifugation and immediately mixed (1:1) with 2× gel sample buffer [0.1 M, Tris-Cl (pH 6.8), 1% (wt/vol) sodium dodecyl sulfate (SDS), 2% (vol/vol) beta -mercaptoethanol, 20% (vol/vol) glycerol, and 0.025% (wt/vol) pyronine Y]. An aliquot of each sample was saved for protein quantification (18).

PKC protein content and activity. Pan-PKC Western blot analysis and PKC activity assays were performed as previously described (25). The pan-PKC antibody recognizes PKC-alpha , PKC-beta , and PKC-gamma . Because we could not detect PKC-beta or PKC-gamma protein, the results obtained with this antibody correspond to those found with the PKC-alpha -specific antibody. Activity assays that measure the phosphorylation of histone III by [gamma -32P]ATP in the presence and absence of phosphatidylserine were performed as previously described (22). The histone substrate does not distinguish among PKC isozymes.

PKC isozyme quantitation. Equal amounts of cytosolic protein (100 µg/lane) were applied to 7.5% SDS-PAGE. Separated proteins were electroblotted onto Immobilon-P membrane, and detection of PKC-alpha , PKC-delta , and PKC-varepsilon with specific antibodies was performed as previously described (6). PKC-alpha bands were quantified by densitometry; A/J and B6 lung samples were run on each gel to serve as samples for normalization.

Northern blot analysis. Lungs were harvested from 6- to 8-wk-old A/J and B6 mice, and total RNA was prepared with the RNeasy kit (QIAGEN, Valencia, CA). Before the cell lysate was loaded onto the spin column, debris was removed by centrifugation in a microfuge at 16,000 g for 5 min. Where applicable, poly(A)-selected RNA was prepared from total RNA with the Oligotex mRNA Mini Kit (QIAGEN). For each sample, 100 µg of total RNA were used as starting material in isolating poly(A) RNA. Northern blots were performed as previously described (36). The blots were probed with a 531-bp EcoR I cDNA fragment of bovine brain PKC-alpha (pbPKC-alpha 21) labeled with [alpha -32P]dCTP to a specific activity of 108 counts · min-1 · µg-1 with the RadPrime kit (GIBCO BRL, Life Technologies, Gaithersburg, MD). The blots were hybridized overnight at 60°C in 15 ml of prehybridization buffer containing 50-75 ng of denatured labeled probe, washed twice at 60°C in a solution of 2× saline-sodium citrate (SSC; 0.15 M NaCl and 15 mM sodium citrate)-0.1% SDS for 30 min, once in 1× SSC-0.1% SDS for 30 min, and once in 0.2× SSC-0.1% SDS for 15 min. Blots were exposed to X-OMAT AR film with an enhancing screen, and the results were quantified by densitometry and with a phosphorimager.

RI mapping. The quantitative traits, lung PKC-alpha content and PKC activity, were mapped with Map Manager QT version 27 and the database for typed loci named A×B and B×A QT (35a). This database has been edited to remove errors, extinct strains, questionable loci, sister strains, and loci with identical strain distribution patterns that interfere with permutation tests of significance (34). To determine whether the likelihood ratio statistics (LRSs) were suggestive, significant, or highly significant, 1,000 permutations were performed. The logarithm of odds (LOD) score can be approximated by dividing the LRS by 4.6.


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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Strain variations in PKC. PKC protein in A/J and B6 lung tissue extracts was quantified in 16 independent analyses by subjecting autoradiograms (pan-PKC antibody) and chemiluminescence films (PKC-alpha -specific antibody) to densitometric analysis. Adult B6 lungs had 2.5-fold more PKC-alpha protein than adult A/J lungs, and B6 mice had 1.6-fold more PKC activity than A/J mice. Twenty other strains were surveyed for their pulmonary PKC activity and contents (Table 1). Only A/J mice had both low PKC activity and content; most strains exhibited both higher PKC activity and content than A/J mice. The correlation between PKC activity and PKC-alpha content in these strains was not significant (r2 = 0.11; P > 0.05). Particulate fractions prepared from A/J and B6 mouse lung tissues reflected the same relative difference in PKC activity and content (when detectable), but there was much less PKC in the particulate fraction; hence, we focused on soluble PKC.

                              
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Table 1.   Strain survey of pulmonary PKC-alpha content and PKC activity

Pulmonary PKC isozyme protein content in A/J and B6 mice. A/J and B6 pulmonary PKC isozyme distributions were determined. Our laboratory previously established that the major isozymes in whole CBy lung extracts (26) and in lung epithelial cell lines derived from A/J, CBy, BALB/c, and Swiss-Webster mice (6) were PKC-alpha , PKC-delta , and PKC-zeta . These proteins were indeed detected in soluble and particulate fractions from A/J and B6 lung extracts (Fig. 1), whereas PKC-beta , PKC-gamma , and PKC-varepsilon were not (data not shown). The multiple bands in the immunoblots for both PKC-delta and PKC-zeta may reflect differentially phosphorylated species or are a consequence of limited proteolysis. More PKC-alpha was contained in B6 lungs than in A/J lungs, whereas no strain differences were detected in PKC-delta and PKC-varepsilon protein contents.


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Fig. 1.   Immunoblot of protein kinase (PK) C-alpha , PKC-delta , and PKC-varepsilon proteins in lung extracts obtained from A/J (A) and C57BL/6J (B6; B) mice. Equal amounts of protein from A/J and B6 lung cytosols were subjected to SDS-PAGE, transferred onto nitrocellulose, and immunoblotted with anti-PKC-alpha , PKC-delta , and PKC-zeta antibodies. Colorimetric detection yielded the ~80-kDa bands shown.

Pulmonary PKC-alpha mRNA content in A/J and B6 mice. To determine whether these differences in protein expression reflected differential mRNA contents, Northern blot analysis was performed on RNA samples prepared from A/J and B6 mouse lungs. Long exposure of film showed threefold more PKC-alpha mRNA in B6 than in A/J lungs; this was true whether total RNA or poly(A)-enriched mRNA fractions were used (Fig. 2). Whether the primary reason for this low PKC-alpha mRNA level in A/J lungs resulted from decreased mRNA stability or reduced transcription of the PKC-alpha gene is not known.


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Fig. 2.   Northern blot of PKC-alpha mRNA in lung extracts obtained from A/J and B6 mice. Poly(A) RNA was isolated as described in METHODS, probed with PKC-alpha probe, and examined with Northern blot analysis. Two PKC-alpha mRNA species were observed at 7.6 and 3.4 kb, similar to those in other mouse organs. A: mRNA from A/J and B6 lungs. Nos. at right, no. of bp. B: quantitation of relative amounts of PKC-alpha mRNA as determined from a Northern blot with total RNA. Equal amounts of RNA were loaded as estimated by absorbance at 260 nm and ethidium bromide staining. Autoradiograms of the total RNA blots were too faint to reproduce photographically. PKC-alpha mRNA content in A/J mouse lung is significantly less than that in B6 mouse lung.

Inheritance pattern of PKC-alpha content based on Mendelian crosses. To determine the mode of pulmonary PKC inheritance, we crossed A/J and B6 mice and examined progeny from F1, F2, and both backcrosses. Lungs were harvested, and their PKC-alpha contents were determined (Table 2, Fig. 3). PKC concentration in the (A×B6)F1 mice is intermediate to that in the A/J and B6 parents, analogous to the intermediate PKC content of lungs from (A × CBy)F1 measured previously (22). Most backcross progeny of the (A×B6)F1 × A backcross have values resembling the A/J parent, whereas values of the (A×B6)F1 × B6 backcross are distributed over the range of both parental strains. This pattern is consistent with more than one gene being responsible for the strain difference in PKC-alpha content. In support of this, we compared the variances in the F1 and F2 generations to estimate the number of genes contributing to the differential lung PKC content using the formula n = (m1 - m2)2/8(VF2 - VF1) = (1.0 - 2.9)2/8(0.25 - 0.0009 = 1.8 genes, where m1 and m2 represent the mean values found in the parental strains, VF2 and VF1 represent the variances of the F2 and F1 progeny, and the following assumptions were made: the genes are unlinked, the genes are codominant, and the genes have equal contributions to phenotype (33).

                              
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Table 2.   Lung PKC-alpha content of A/J and B6 parental strains and F1, F2, and backcross mice



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Fig. 3.   Inheritance patterns of PKC-alpha content in A/J and B6 lungs. Relative PKC-alpha contents in lung cytosolic fractions from A/J (A), B6 (B), (A×B6)F1 (AB F1; C), (A×B6)F2 (AB F2; D), A/J × F1 backcross (E), and B6 × F1 backcross (F) mice were determined and normalized to A/J value. Each symbol represents an individual mouse; n, no. of mice.

A×B and B×A RI analysis of PKC-alpha content and PKC activity. PKC-alpha content and PKC activity in the A×B and B×A RI strains were determined, and QTL analyses were performed; PKC activity and PKC-alpha content in these RI strains correlate well (r2 = 0.74; P < 0.005; Table 3). Lung PKC-alpha content and PKC activity were each entered into Map Manager QT as quantitative traits with the edited database of A×B and B×A prepared especially for quantitative trait analysis (34). The major locus for PKC-alpha content maps to chromosome 11, with the highest peak at D11Mit333 (P < 0.0004; Fig. 4). This is the same location as the PKC-alpha structural locus, although PKC-alpha has not been previously mapped in these RI strains. The LRS for this map position is 12.5, which is approximately equal to a LOD score of 2.7. Permutation tests give LRSs of 4.9, 15.3, and 24.8 as suggestive, significant, and highly significant, respectively. A suggestive peak with a LRS of 6.2 at D3Mit19 was found as well. The major locus for lung PKC activity (Fig. 5), on the other hand, maps to chromosome 3 at D3Mit19 with an LRS of 15.4 (P < 0.00008; LOD = 3.4). Permutation tests give LRSs of 5.2, 14.2, and 25.0 as suggestive, significant, and highly significant, respectively. Additionally, there is a suggestive peak for PKC activity with a LRS of 5.1 at D11Mit333.

                              
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Table 3.   Lung PKC-alpha activity and content of A×B and B×A RI strains relative to A/J strain



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Fig. 4.   Quantitative trait locus (QTL) analysis of lung PKC-alpha content. Lung PKC-alpha content was determined in 23 A×B and B×A recombinant inbred strains and normalized to A/J value. These values were used in Map Manager QT to locate the major locus determining this trait.



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Fig. 5.   QTL analysis of lung PKC activity. Lung PKC activity was determined in 23 A×B and B×A strains and normalized to A/J value. These values were used in Map Manager QT to locate the major locus determining this trait.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Of 22 inbred strains examined, only A/J mice had both low PKC-alpha content and PKC activity (Table 1). The inheritance pattern of PKC-alpha content elucidated with F1, F2, and backcrosses with both A/J and B6 parents suggests that two genes are responsible for these strain variations (Table 2, Fig. 3). Although the correlation between PKC-alpha content and PKC activity among inbred strains was not quite significant, content and activity among the A×B and B×A RI strains did correlate with each other (r2 = 0.74; Table 3). The major determinant of PKC-alpha content mapped near the PKC-alpha structural gene at 68 centimorgans on chromosome 11, with a minor modifying gene that influenced PKC-alpha content at D3Mit19 or D3Mit94 (Fig. 4). The major determinant responsible for the low PKC activity in A/J mice mapped at 88 centimorgans on chromosome 3, with a minor determinant at the PKC-alpha structural gene locus Pkca (Fig. 5). Because the basis of the low PKC-alpha in A/J mice is a reduced level of PKC-alpha mRNA (Fig. 2), the chromosome 11 site responsible for this reduction could be the PKC-alpha promoter sequence, with the chromosome 3 site encoding a transcription factor that binds differentially to the A/J versus B6 PKC-alpha promoters. Alternatively, A/J mice might have a polymorphism in the 3'-end of the message that increases its interaction with ribonucleases, with the chromosome 3 site encoding a protein that regulates mRNA stability by binding to this 3' site.

The developmental expression of different PKC isozymes is strain, isozyme, and tissue specific (38). An analogous situation to what we observed with PKC-alpha in lung has been reported with hippocampal PKC-gamma . Within the hippocampus, the concentrations of six PKC isozymes increased postnatally, whereas four others did not (15). A strain dependence in the postnatal timing of PKC-gamma was observed wherein neonatal B6 and DBA mice have similar PKC-gamma content 10 days after birth, but enzyme content in B6 mice increased to a greater extent with age than in DBA mice (30). This has been associated with the deficit in complex learning and memory that DBA mice display relative to B6 mice. A combination of deletion analysis and DNase footprinting was used to examine the developmental expression of rat brain PKC-gamma (1). A 23-bp sequence within the 5' region was bound by neonatal nuclear proteins to a greater extent than it was by nuclear proteins from adult rat brains. Because PKC-gamma expression increased with age, nuclear proteins that bound to this site correspondingly decreased. Thus a cis-acting site repressed expression.

The basis of developmental regulation of the rate of protein synthesis among different cell types is not understood (3). Information has been obtained with transgenes consisting of a reporter construct attached to a host gene promoter. The degree to which transgene expression corresponds to expression of the endogenous gene with regard to cell specificity and age depends in part on the amount of ligated 5' sequence and whether any 3' or intronic sequence is also needed. A cis-acting site close to a structural gene plus another that may act cis or trans (i.e., reflecting the need for a diffusible product) is often required for normal ontogenic appearance. For example, if the 5' sequence ligated to a Clara cell-specific reporter was too short, the transgene was expressed prematurely during development (14).

The phenotype of an advanced tumor resembles its tissue of origin at an earlier developmental stage more than it does adult tissue (37). This is illustrated in human lung cancer where the enzymatic content and isozyme distribution of dozens of enzymes were more analogous to fetal lung tissue than to adult tissue (13). Mouse lung tumors have the low PKC-alpha content that is more characteristic of neonatal lung than of adult lung (32). Genes that regulate the organ-specific appearance during development of an enzyme involved in neoplastic conversion seem reasonable candidates for susceptibility genes. One of the fourteen genes that regulate susceptibility to lung tumorigenesis in mice is near the chromosome 11 site that regulates the postnatal appearance of PKC-alpha (10). How might the low PKC-alpha content in A/J mice contribute to their enhanced tumor susceptibility? The lungs of A/J mice are unique in their exquisite sensitivity to induced and spontaneous tumorigenesis (21), infection (16), acute injury (31), and airway hyperactivity (2). Tumor-promoting agents such as phorbol esters and butylated hydroxytoluene decrease PKC content in vitro (5), and pulmonary PKC-alpha content decreased 30 min after butylated hydroxytoluene administration to mice (26). Because high PKC-alpha levels are associated with a differentiated, proliferatively quiescent phenotype in mouse lung epithelium (6), low intracellular PKC-alpha concentrations might predispose mice toward proliferative disorders such as neoplasia.


    ACKNOWLEDGEMENTS

We thank Cathy Auerbach and Kat Keil for excellent technical assistance.


    FOOTNOTES

This work was supported by a Parker B. Francis Fellowship (to L. D. Dwyer-Nield) and National Cancer Institute Grant CA-33497 (to A. M. Malkinson).

Address for reprint requests and other correspondence: A. M. Malkinson, Dept. of Pharmaceutical Sciences, School of Pharmacy, UCHSC, C-238, 4200 E. 9th Ave., Denver, CO 80262 (E-mail: Al.Malkinson{at}UCHSC.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 10 November 1999; accepted in final form 29 March 2000.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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