Mitogen effects on lipid metabolism during lymphocyte activation

Molecular Immunology, Vol. 20, No. 12, pp. 1259-1265, Printed in Great Britain.

MITOGEN

Guelph-Waterloo

1983

0161.5890183 $3.00 + 0.00 Pergamon Press Ltd

EFFECTS ON LIPID METABOLISM LYMPHOCYTE ACTIVATION

DURING

DOREEN NATHANIEL* and ALAN MELLORS~ Centre for Graduate Work in Chemistry, University of Guelph, Ontario. Canada NIG 2Wl (Received 8 March 1983; accepted 25 April 1983)

Abstract-Mitogenic and non-mitogenic lectins have been compared for their abilities to stimulate acetate and choline incorporation into mouse spleen lymphocyte lipids. The concns required for maximal acetate incorporation correspond to those required for maximal blastogenesis. The non-mitogenic lectins tested had no effect on acetate incorporation. Concanavalin A also stimulated acetate incorporation into splenic lymphocyte lipids of athymic mice which do not undergo T-cell blastogenesis. 4,4’-Diaminodiphenylsulfone (dapsone) at 150~g/ml inhibited the incorporation of choline into mouse spleen lymphocyte phosphatidylcholine by 40x, but had no significant effect on concanavalin A induced mitogenesis when the inhibitor was present during the first few hours of transformation. Enhanced turnover of phospholipids appears to be a parallel but non-essential event in the early stages of mitogenesis.

INTRODUCTION Resting lymphocytes can be induced to become metabolically active, to differentiate and divide, upon treatment with a mitogen. Binding of the mitogen to its receptor on the lymphocyte membrane results in a variety of metabolic and morphologic changes, some of which occur within minutes of the addition of the mitogen, and are believed to be associated with the transmission of a signal for cellular proliferation (Hesketh, 1978; O’Brien et al., 1978). The relationship between many of these early events and the later occurrence of DNA synthesis remains obscure. Turnover of phosphatidylinositol (PI), a minor component of the membrane phospholipids, is greatly enhanced within minutes of the addition of a mitogen such as phytohaemagglutinin (PHA) (Fisher and Mueller, 1968, 1971). Increased synthesis and turnover of phosphatidylcholine (PC), the major membrane phospholipid, is stimulated less rapidly, from 1 hr and continuing up to 68 hr after addition of the mitogen (Kay, 1968; Fisher and Mueller, 1969). More marked however is the turnover of fatty acyl chains of PC and other phospholipids, believed to greatly exceed de nouo PC synthesis as an early event in lymphocyte activation (Resch and Ferber, 1972). One model for lymphocyte activation suggests that this rapid turnover of phospholipid acyl chains is due to activation of the enzyme acyl-CoA: l-acylglycero3-phosphocholine O-acyltransferase (ATase) (EC 2.3.1.23), with a resultant increase in membrane fluidity. The increased membrane fluidity, it is proposed, may be a signal for cellular proliferation

*Present address: Merck, Frosst, Canada Inc., P.O. Box 1005, Pointe Claire, Dorval, Quebec, Canada H9R 4P8. tTo whom correspondence should be addressed.

(Ferber and Resch, 1973; Resch, 1976). Although there is evidence that in intact lymphocytes ATase is not activated in mitogenesis, the rapid turnover of phospholipids has been confirmed (Dobson and Mellors, 1980). Enhanced phospholipid turnover may be part of an overall increase in synthesis or turnover of membrane lipid components. In this report we compare early changes in lipid metabolism and a parameter of blastogenesis, enhanced DNA synthesis. Evidence reported here shows that early enhanced turnover of phospholipid acyl chains in mouse spleen lymphocytes is a response which can result from interaction of T-cell mitogens with lymphocytes from normal and athymic (nude) mice. T-Lymphocytes from athymic mice do not undergo blastogenesis when exposed to T-cell mitogens (Andersson et al., 1972). An inhibitor of de nouo synthesis of PC has been used here to inhibit choline incorporation into lymphocyte phospholipids and the results suggest such inhibition does not interfere with lymphocyte transformation by mitogens. In studies which compare the incorporation of acetate into lymphocyte phospholipids following exposure to various lectins, we conclude that the enhanced turnover of acyl chains is a general response of cells to the binding of specific lectins, and is a parallel but non-essential event in lymphocyte activation by these agents. MATERIALS

AND METHODS

Chemicals

Concanavalin A valin A (S-Con A), tive of Con A (Wang Vector Laboratories N-acetylglucosamine

1259

(Con A) and succinyl-concanathe divalent succinylated derivaet al., 1976), were obtained from (Burlingame, CA); other lectins, and 4,4’-diaminodiphenylsul-

DOREENNATHANIEI L and ALAN MELLOKS

1160

fone (dapsone) were from Sigma Chemical Co. (St. Louis, MO); and cell culture media were from Gibco (Burlington, ON).

as a measure of the level of DNA synthesis (mrtogenesis) occurring in the cell. Lectrns. in 50 /II HBSS. were added to lO”cells in 1 ml RPM1 containing sodium bicarbonate, IO”, fetal calf serum, and 50 pg/ml gentamycin sulphate. Lymphocytes were then incubated for 46 hr at 37 C in a humidified 5”,, CO, atmosphere. Cell viability at this time. as determined by trypan blue exclusion, was 70 7S”,,. One microcurie of [‘Hlthymidine was added to each tube. and the incubation was continued for a total incubation time of 48 hr. Cells were harvested on filter papers in a Titertek cell harvester (Flow Laboratories). Filters were washed thoroughly with 5”; TCA, dried and placed in scintillation vials to which 5 ml scintillation fluid was added (15 g 2,5-diphenyloxazole in 3.8 I of toluene). ‘H-Radioactivity was counted in a Beckman LS7000 liquid scintillation counter.

[ I-‘%Z]Acetate as the sodium salt (58 Ci/mole) and [methyl-‘4C]choline chloride (54 Ci/mole) were obtained from New England Nuclear (Lachine, QU); [6-‘Hlthymidine was obtained from Amersham Corp. (Oakville, ON) (5 Ci/mole). Lymphocyte isolution. White Swiss-Webster mice (Connaught Laboratories strain or OVC inbred colony), male and female, 8 weeks old, were obtained from the Animal Laboratory Facilities, University of Guelph. Congenitally athymic nujnu (nude) mice, RNC line, male or female, 6-8 weeks old, were obtained from Brian Bloomfield, Clinical Studies, University of Guelph. Spleens were excised and gently hand-homogenized in a Teflon-glass homogenizer with IOml Hanks’ balanced salt solution (HBSS), pH 7.4. The suspension, obtained after allowing connective tissue and fat to settle out from the homogenate, was centrifuged at 400g for 10 min. The pellet was cleared of intact red blood cells, dead cells and platelets by hypotonic lysis according to the method of Boyle (1968). The cell pellet obtained after the last step was resuspended in either RPM1 1640 with 25mM HEPES and L-glutamine at a concn of IO’ cells/ml for short-term cultures, or in RPM1 1640 with 5 mM sodium bicarbonate and L-glutamine at a concn of lO”cells/ml for long-term cultures. Cell concns were determined using a hemacytometer and lymphocyte viability was measured by trypan blue exclusion. Only preparations of greater than 90% viability were used for culturing.

[‘“Cl Acetate incorporation into lymphocyte lipids Lymphocyte lipid metabolism was studied by following the incorporation of the precursor [‘“Clacetate into several lipid fractions [lysophosphatidylcholine (LPC), PC, phosphatidylethanolamine (PE), fatty acids (FA) and triglycerides (TG)]. To IO’cells in RPM1 1640 with HEPES was added 25 nmoles [14C]acetate (I .4 FCi) in 50 ~1 HBSS, followed immediately by 50 ~1 HBSS containing lectin. The mixture was incubated for 4 hr in a 37°C shaking water bath. The reaction was terminated by the addition of 2 ml cold TCA, and the mixture centrifuged at IOOOg for IOmin. The lipids were extracted from the TCA precipitate according to the method of Bligh and Dyer (1959) and isolated by chromatography on thin layers of silica gel (Dobson and Mellors, 1980). Radioactivity was determined in each fraction by liquid scintillation counting using Anderson’s scintillation fluid: xylene (75”:, v/v), Triton X-100 (25:~” v/v), diphenyloxazole (0.3”,, w/v).

[‘HI Thymidine uptake The uptake of [3H]thymidine chloroacetic acid (TCA) insoluble

Table

I.

mouse

into cellular trimaterial was used

Effects of l&ins

spleen lymphocyte

and hpopolysaccharide phosphatidylcholine dine

on [‘4C]acetate (PC)

compared

incorporation to the elkcts

in PC Agent A

(cpm)

5650 + 200 13,000 + 350* A

5700 * 400 8000 f

Lrns

rulinaris

&tin

200*

2500 * 400

1000’

9000 * Pi.wn

lectin

sa1i1wn

600 f

640

15,000 f 900’ E. colr lipopolysaccharlde Wheat

germ

011

615Ok

150

50

6050 i

40

0

5650 i

400

acetate

hFor

thymidine

‘Values

I600 42,000

+ 300 k 3000’

1600 k 300 22,500

i_ I lOO*

400 ? 250 67,000

* 7000*

115oi 32.800

150 i

4350 i

3000* 800

I :B4

agglutinin

mcorporation,

5100 + 450

140 *

3700 + 80

1160+40

3800 + 170

n = 4. Results

different

from

are expressed

controls

at P < 0.05.

as f SEM

1500*

1220 k 60

0

n = 3.

incorporation,

are significantly

17.250 i

16 16 “For

[‘H]Thymidim? incorporation

&Pm)

Succmyl-concanavalin

[‘Hlthymi-

incoruoration [‘4C]Acetate”

Concanavalin

into Swiss-Webster on cellular

lo*

990 + 100

1261

Lipid metabolism and lymphocyte activation [‘T]Choline

incorporation into PC

De nova synthesis of PC was followed by the incorporation of [‘4C]choline in the presence of lectins or the non-toxic choline incorporation inhibitor, dapsone. The dapsone in 5 p1 DMSO or lectin at the appropriate concn in 50 ~1 of HBSS, was added to IO’ cells in 1 ml RPM1 1640, with 25 mM HEPES and L-glutamine in 1.5-ml microcentrifuge tubes. Immediately after lectin or dapsone addition, 1 PCi labelled choline was added in HBSS and the mixture was incubated for 4 hr in a 37°C shaking water bath. Dapsone-treated cells were checked for viabilities using the chromium release assay (Sullivan et al., 1972). The incorporation of choline was stopped after the incubation period by addition of 0.1 mg unlabelled choline and then centrifuging at 13,OOOg,,, for 30 set on a Beckman Microfuge B. The supernatant was removed and the lipid-containing pellet was washed twice with phosphate-buffered saline to remove unincorporated labelled choline. The pellet was then extracted according to the method of Bligh and Dyer (1959), with 0. I mg dipalmitoylphosphatidylcholine added to the extracting solution to help in the recovery of the radioactive PC. This extraction was repeated twice, the supernatants pooled and 1 ml SOo/, v/v aqueous methanol was added to the supernatant to form a two-phase system. The aqueous layer was removed and the chloroform layer containing PC was washed with 1 ml water. An aliquot (0.5 ml) of the chloroform layer was put in a scintillation vial and dried down under N,, 0.5 ml methanol was then added with 5 ml Anderson’s scintillation fluid and “C-radioactivity was determined by scintillation counting.

2 and 4 pg/ml, and was close to maximal at 8 pg/ml. Acetate incorporation into PC was maximal at 8pgg/ml of P. sutivum lectin and remained close to maximal at 16 pg/ml. All the T-cell mitogens stimulated PC turnover, but LPS, the B-cell mitogen, had no effect on acetate incorporation. The non-mitogenic lectins (Goldstein and Hayes, 1978), wheat germ agglutinin (WGA) specific for N-acetylglucosamine, and Dolichos biforus agglutinin specific for N-acetylgalactosamine, had no effect on acetate incorporation.

Con A

RESULTS

the B-cell mitogen E. colt’ lipopolysaccharide (LPS) were tested over a range of concns for their ability to stimulate [14C]acetate incorporation into mouse spleen lymphocyte lipids, and for their blastogenic effectiveness. The lipid classes PC, phosphatidylaminoethanol (PE), LPC, FA and triacylglycerols (TG) were monitored for acetate incorporation but only the PC results are shown in Table 1. The lectin concns shown in Table 1 are those that gave both maximal stimulation of acetate incorporation and rH]thymidine incorporation in parallel experiments. A typical pair of dose-response curves for one of the mitogens tested, Con A are shown in Fig. 1. Con A produced maximal stimulation of thymidine incorporation at a concn of 2 pg/ml with an abrupt fall-off of incorporation with Con A concns higher than 2 pg/ml. For all mitogens except Pisum sativum lectin, the concns which gave maximal thymidine incorporation coincided with those which gave maximal acetate incorporation into all lipids tested. Thymidine incorporation in lymphocytes treated with P. sativum lectin was maximal between Six lectins

pglml

B

and

60

40

20

20

10

0 4 6 12 16 Fig. 1. Effects of Con A on: (a) acetate incorporation into lymphocyte lipids: (0) PC, (0) TG, (m) PE, (0) FA and (A) LPC. The error bar shows the S.E.M. only when it exceeds 10% (n = 3). (b) Thymidine incorporation as a measure of blastogenesis: (0) Swiss-Webster mice; (A) athymic mice (n = 4).

DOREEN NATHANIEL and ALAN M~LI.ORS

26

.

. 24

.

.

t

v

v

1 -

0 1-

0

3

4

6

6

10

12

14

16

Leclin pg ml-’

0

0

0

.I

2

0

.

.

4

.

12

a

16

Con A pglml

Fig. 2. Acetate incorporation into lipids of lymphocytes from athymic mice: effect of Con A. (m) PC, (V) TG plus FA, (0) PE and (A) LPC. The error bar shows the S.E.M. only when it exceeds

loo/, (n = 3).

Splenic lymphocytes from athymic mice do not undergo blastogenesis when treated with the T-cell mitogen, Con A (Andersson et al., 1972). In this study, Con A was shown to stimulate lipid turnover in both normal and athymic mouse spleen lymphocytes. Figure 1 shows the effect of Con A on acetate incorporation into lipids of Swiss-Webster mouse spleen lymphocytes. Acetate incorporation into these lipids is maximally elevated at the optimal mitogenic concn of Con A, 2 mg/ml, and is lower at the higher concns, as described by other workers (Dobson and

Fig. 3. Choline incorporation into lymphocyte lipids: efccts of increasing lectin dose. Choline uptake is expressed as percentage of control values: (0) Con A, (A) S-Con A, (0) WGA. The error bar shows the S.E.M. only it exceeds 10%. Control values for Con A: 1007, = 20,000 & 300cpm (n = 3); S-Con A: lOO”d,= 40,500 f 1000 cpm (n = 3); and WGA: 100% = 44,800 f 800 cpm (n = 3). Significant differences were found for all Con A-treated samples (P < O.OS), 8 pg/ml S-Con A (P < 0.1) and 16 pg/ml S-Con A (P < 0.01).

Mellors, 1980). Figure 2 shows comparable data for lymphocytes from athymic mice. Maximal incorporation was seen at a higher concn of Con A, 8 pgg/ml, than in normal lymphocytes. Stimulation of acetate incorporation in the presence of Con A was about half that seen in normal mouse lymphocytes. Again a reduced acetate incorporation response was seen at high lectin concns. In normal and athymic mouse lymphocytes, the largest increases are seen for the turnover of PC and PE. All T-cell mitogens which stimulated acetate incorporation into PC in normal

Table 2. Etkcts of l&ins and lipopolysaccharide on [‘4C]acetate incorporation into athymlc mouse spleen lymphocyte phosphatidylcholine (PC) compared to the effects on cellular [3H]thymidine incoraoration COllCll

Agent Concanavalin Succinyl-concanavalin Lens culinaris lectin Pisum mlrc‘um lectin E. co/i Ilpolysaccharide 0111: B4 Wheat germ agglutinin Dolrchos biflorus lectin

A

[‘4C]Acetate” in PC

bs/mU

@Pm)

0 8 0 32 0 16 0 16 0

18OOi40 2900 f 220* 3900 f 190 6500 + 450* 1250 f 40 2100 i_ 160* 4100 i 40 6170 f 160* 1500~50

SO 0 16 0 16

1800 f IO* 2000 * 130 2100 * 120 175Ok80 1700+50

“For acetate incorporation, n = 3. *For thymidme incorporation, n = 4. Results are expressed as + SEM *Values are significantly different from controls at P < 0.05.

[‘H]Thymldineh incorporation (cpm) 730 400 580 330 550 160 500 200 520 23.460 630 550 540 570

i * i * * + f + f

50 40 30 20 30 IO 30 30 30

+ 480* + 40 * IO & 30 + 30

Lipid metabolism and lymphocyte activation IO5

1263

+ con A T

0

d t ::

IO4

B 8 t ._

8

t

c” 7

20

I

K

E I

1

I

1

I

50

100

150

200

250

$

102

Osprone )rg/ml

Fig. 4. Inhibition of choline incorporation into lymphocyte lipids by dapsone. Choline incorporation is expressed as percentage of control values. Hundred per cent control represents 62,000 f 4600cpm. The error bar shows the S.E.M. for each point (n = 3). mouse lymphocytes exerted a similar effect on the athymic mouse lymphocytes but to a lesser extent, as shown by comparing Tables 1 and 2. It should be noted that the stimulation in athymic mouse lymphocytes occurs at concns much higher than those giving similar results in normal mouse lymphocytes. The B-cell mitogen, LPS, showed a mild stimulation of acetate incorporation into PC of athymic mouse spleen lymphocytes but did not affect acetate incorporation in normal mouse spleen cells. The stimulation of the incorporation of choline into PC, on the binding of mitogenic lectins, has been implicated in lymphocyte activation (Chen, 19790) and we have compared this increased de nova synthesis of PC with the acyl chain turnover as measured by acetate incorporation studies. Figure 3 shows the effect of three lectins on choline incorporation into mouse spleen lymphocytes. A comparison of the dose-response curves for Con A, S-Con A and WGA shows that only Con A causes a great enhancement of choline into PC. The maximal stimulation of incorporation occurs at a Con A concn of 8 pg/ml. There is a slight enhancement of choline incorpor-

Table 3. Washing

does not overcome

Controls (no dapsone)

12hr @3=-)

7100 * 50 28,500 + 900

I

I

I

1

0

50

100

150

I

200

I 250

Daprono ~glml

Fig. 5. Effect of dapsone on lymphocyte blastogenesis. Lymphocytes were incubated with Con A and/or dapsone for 4 hr. Dapsone was washed from the cells and the incubation continued to 48 hr. (0) Lymphocytes treated with Con A and dapsone; (B) lymphocytes treated with dapsone only. The S.E.M. is represented for all points (n = 4).

ation in the presence of S-Con A at higher concns (8 and 16 hg/ml). There appears to be no effect of WGA on choline incorporation over the range of concns tested. An inhibitor was used to examine whether elevated choline incorporation into PC was important for the later event of lymphocyte transformation. Dapsone is an inhibitor of choline incorporation and is relatively non-toxic (Shigeura et al., 1975). If PC synthesis is limiting for transformation, then inhibition of PC synthesis should lead to cells with impaired ability to transform in the presence of a mitogen. Dapsone inhibited choline incorporation by 50% at a concn of 250pg/ml (Fig. 4). Cell viabilities were checked at

the effect of dapsone

Choline Incubation period

102

on [“C]choline

mcorporation

incorporated per IO’ lymphocytes @pm, mea” k SEM”) 250 @g/ml 40 pg/ml dapsone dapsone 6750 f 190* 27,700 i 600

4470 * 500** 17.700 i: 4350”

(n = 3) “Lymphocytes (10’ml RPM1 1640 with 5% FCS) were incubated with dapsone for 4 hr, at which time cells were washed once with PBS and then re-incubated in fresh medium for time period indicated. Addition of 1 pCi [‘4C]choline to the cell culture was made in the last 4 hr of the incubation period. *Significantly different from control values at P c 0.05. **Significantly different from control values at P c 0.01.

I264

DOREEN NATHANIEL and ALAN MBLLOKS

various dapsone concns to see if the decreased choline incorporation was due to cell death. At concns up to 250pg/ml, cell viabilities were still high (85”! at 250 ng/ml). Dapsone effects on transformation were examined by incubating cells with lectin and dapsone for 4 hr. All cells were washed with PBS to remove dapsone and then incubated as usual for the assay for blastogenesis. Cells treated with dapsone have an impaired ability to synthesize PC but do not show significant impairment of DNA synthesis at 48 hr. It is apparent from Fig. 5 that low levels of dapsone cause a slight elevation of [3H]thymidine incorporation in the absence of mitogen but the reasons for this increase in basal DNA synthesis are not clear. The removal of dapsone by washing with PBS was not complete, and Table 3 shows that there is still some impairment of choline incorporation at 43 hr when high concns of dapsone were used. However blastogenesis was unaffected by the residual dapsone, and by the impaired de nouo synthesis of PC. DISCUSSION It is known that T- and B-lymphocytes bind T-cell mitogens to the same degree (Goldstein and Hayes, 1978). A major determinant for the specificity of activation by a mitogen is not the degree of binding but the extent of cross-linking of specific receptors on the cell surface (Greaves and Janossy, 1972). The alterations in lipid metabolism upon binding of mitogens appears to be much less specific. The absence of certain early metabolic events in lymphocytes of athymic mice upon binding of T-cell mitogens, has been used as evidence of a causal relationship between these early events and subsequent DNA synthesis (Owens and Kaplan, 1980; Freedman and Khan, 1979). There have been few reports concerning the effects of T-cell mitogens on lipid metabolism in lymphocytes of athymic mice, though a brief report suggested that Con A did not stimulate oleate incorporation into PC in athymic mouse lymphocytes (Resch et al., 1976). Similarly Betel and van den Berg (1975) reported no increase in acetate incorporation into phospholipids of athymic mouse lymphocytes after 24 hr of exposure to Con A. The discrepancy between those results and the results presented here may possibly be explained by the differences in the time of measurement. Also the Con A concn, 8 pgg/ml, required for maximal stimulation of acetate incorporation in athymic mouse lymphocytes is higher than for normal mouse lymphocytes (Tables 1 and 2). A concn adequate in normal lymphocytes for stimulation of acetate incorporation is not adequate in lymphocytes of athymic mice. Increased lectininduced acetate and oleate incorporation into PC of athymic mouse lymphocytes has been reported, though this was attributed to contamination with pre-T cells (Resch and Bessler, 1981). That immature T-lymphocytes are capable of showing increased early synthesis or turnover of PC has been shown by

Pratt er al. (1977), who measured incorporation of [Ylcholine into phospholipids of human thymocytes incubated with Con A or PHA for 6 hr. However lymphocytes of athymic mice are reported to contain only a few percent of immature T-lymphocytes (Janossy and Greaves, 197 I ; Milich and Gershwin, 1977). Our results support the idea that the increased turnover of fatty acyl chains occurs when a T-cell mitogen binds to spleen lymphocytes, whether from normal or athymic mice, and that this event is not necessarily associated with blastogenesis. Although it has been recognized that early de no{:0 synthesis of PC during mitogenesis occurs more slowly than the turnover of the acyl chains, this event has been linked with the later occurrence of DNA synthesis. For example, Chen (1979~ ) has reported that increases in PC and PI synthesis proportional to Con A concns are pre-commitment events since the increases can be abolished with the addition of r-methyl mannoside (a-MM), a competitor for Con A binding. r-MM does not abolish the increased sterol synthesis seen later in blastogenesis (Chen. 19796). The data on choline incorporation presented here reinforces the argument that tie noro synthesis of PC is not enhanced to the same extent as turnover of PC acyl chains. It should be noted, however, that in the present study the optimum Con A concns required for blastogenesis did not produce maximum choline incorporation in Swiss--Webster mouse spleen lymphocytes. Early de nova synthesis of PC during lymphocyte blastogenesis is inhibited by dapsone. Previous reports showed that dapsone was a relatively non-toxic inhibitor of choline incorporation into PC in macrophages (Shigeura et cd., 1975; Bonney et al.. 1979). The effects of dapsone on choline incorporation in lymphocytes were reversible only at low concns of the drug. With low concns of dapsone, where choline incorporation was inhibited up to 35”A, lymphocyte transformation by Con A was unaffected. Higher concns of dapsone (160 and 250 pg/ml) slightly inhibited lymphocyte blastogenesis. While the binding of T-cell mitogens to mouse spleen lymphocytes produces numerous changes in membrane lipid metabolism there is evidence that these events are not essential for blastogenesis. It is possible to inhibit LPC acyltransferase and choline incorporation without affecting blastogenesis. Increased acetate incorporation is seen in cells which do not undergo blastogenesis when exposed to mitogens. Acknovvledgemmts-We would like to thank M. Sadler and M. Stalmach for expert technical assistance and the Natural Sciences and Engineering Research Council of Canada for financial support.

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MlMM

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1265

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