1

t-PA and plasminogen embedded in casein rule

its degradation

Nissim Silanikovea*, Fira Shapiroa , Gabriel Leitnerb

aa Biology of Laction Lab., Institute of Animal Science, Agricultural Research Organization, The Volcani Center, P.O.

Box 6, Bet Dagan 50250, Israel

b bNational Mastitis Reference Center, Kimron Veterinary Institute, Bet Dagan 50250, Israel

*Corresponding author: email: nsilanik@agri.huji.ac.il

2

Abstract

The aims of this study were to test the assumption that tissue-

plasminogen activator (t-PA) and plasminogen are closely associated to

the casein micelle and form a functional complex that rule casein

degradation. This assumption was essentially verified for bovine’s milk.

It was also shown that the second type of plasminogen activator presented

in milk, urokinase-PA (u-PA), was not involved in casein degradation. It

was found that t-PA and plasminogen are found in freshly secreted milk

(less than 10 min from its secretion), strongly suggesting that they are

secreted as complex by the mammary gland epithelial cells.

Keywords: t-PA, u-PA, plasminogen, plasmin, casein, milk, bovine

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Introduction

Milk secretion rate in various mammals is tightly associated with

the fluctuating offspring nutritional demands and mammary gland (MG)

development stages are closely related to the nurse’s reproduction cycles

[1-2]. The regulation of day-to-day variation in milk secretion [2-3] and

induction of MG involution [4] are ruled by milk-borne negative

feedback (MBNF) system. The plsminogen activator (PA)-plasminogen

(PG)- plasmin (PL) enzymatic system is ubiquitously expressed in the

milk of human [5], rodents [6], and ruminants [4] and was found to be

associated both with regulation of milk secretion in cows and goats [7-8]

and activation of involution in rodents [6], goats [9] and cows [10-12].

As in other body tissues, PL is presented in milk mainly by its

inactive zymogen form PG, whose conversion to PL is modulated by PAs

[12]. The two types of PAs that exist in mammals systemic fluids,

urokinase-type PA (u-PA) and tissue-type PA (t-PA) are also presented in

milk [6,12-14]. In milk, PL, PG, and t-PA are closely associated with the

casein micelles, whereas u-PA is associated with neutrophils in close

association to its specific receptor [12, 14].

The MBNF system associated with regulation of milk secretion

was shown to comprise the PA- PG- PL system that specifically forms a

β-casein (CN) fragment (f) (1–28) from β-CN, which in return, serves as

the negative control signal by closing potassium channels on the apical

membrane of the epithelial cells of the MG [7-8]. Down-regulation of

these channels induces undefined inwardly directed cellular signals that

inhibit milk secretion. Interestingly, a further activation of the PA-PG-PL

system, which was coupled to more extensive degradation of casein

induced involution of the MG in lactating goats and cows and forcefully

activated the innate immune system [9-11]. Based on these findings, a

casein hydrolyzate (CNH) preparation was developed to reduce the

suffering from MG engorgement associated with abrupt cessation of

milking (the conventional procedure to induce involution in modern dairy

cows) [15] and to treat and prevent common clinical and subclinical

infections of the udder in dairy cows [16-18]. It was also found that CN-

derived peptides induced by PL activity inhibits milk clotting [19-20],

which is important during mastitis and milk stasis in preventing

uncontrolled inflammation [21].

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There are convincing evidences that under physiological situations

t-PA is the main factor that involve in casein hydrolysis [6, 12, 22].

However, the picture remained unclear because: i. there is a lot of data

showing that u-PA is involved as a major factor in CN hydrolysis in

stored milk [23], and ii. That the increase of u-PA secretion by MG

epithelial cells under inflammation (12, 22] was claimed to be responsible

for CN degradation in goat’s milk under mastitis [24].

The association of t-PA with the CN micelles suggests that it

constitutes a pivotal component of the MBNF system; however, as noted

above, evidence for the involvement of u-PA confronts this assumption.

The ability of CNH to up regulate the PL system activity in a manner that

imitate in accelerated manner the events associated with MG involution

stage 1 [9-11] represent an opportunity to test the assumption regarding

the pivotal role of t-PA in CN degradation under relevant physiological

situation.

The aims of the present experiment were: i. to test the assumption

that t-PA and PG embedded within casein micelles form a functional

complex that rule casein hydrolysis by analyzing the distribution of the

PL system in different fraction of bovine milk in control and in response

to CNH treatment, ii. to test the assumption that t-PA, PG and CN micelle

are secreted by MG cells as functional unit by verifying the presence of t-

PA and PLG in CN micelle in freshly secreted milk, and iii. to exclude

role of u-PA in CN hydrolysis under the experimental conditions.

5

Material and Methods

Materials. H-D-Norleucyl-hexahydrotyrosol-lysine-pnitroanilide

diacetate [Spectrozyme PL (SpecPL)], bovine PG, and cyanogens

bromide fibrinogen digest (FIBGN) were purchased from American

Diagnostica (Greenwich, CT, USA). N-methylsulphonyl-D-Phe-Gly-Arg-

4-nitroanilide acetate substrate was obtained from Boehringer Mannheim

(Chromozym t-PA; UK, East Sussex, UK). Polyclonal rabbit anti-human

t-PA IgG was obtained from Oxford Biomedical Research (Oxford, UK)

and Plasminogen activator inhibitor-1 (PAI-1), was from Calbiochem

(USA). Other mentioned chemicals were purchased from Sigma

(Rheovot, Israel).

Ethical considerations. All protocols were approved by the Institutional

Animal Care Committee of the Agricultural Research Organization,

which is the legitimate body for such authorizations in Israel.

Experiment 1. Six Israeli Holstein heifers with low leukocyte content, as

indicated by low somatic cell count (< 70,000 cells/ml) and no bacterial

finding according to preliminary analysis [17], yielding ~36 l/day-1

milk,

at their second to third were lactation used. Two MGs, one front and one

rear quarters, were infused with sterile saline solution while the other two

counter were infused with casein hydrolyzate (CNH).

The experiment was carried out during November under natural

lighting regimen, with typical noon temperatures of 24 C0

and night

temperatures of 12 C0, which is within the thermoneutral zone of cows

[25]. The cows were milked thrice daily (0530, 1230, and 2130) and milk

yield and exact milking times were individually recorded automatically

[18].

All the experimental procedures were carried out during the noon

milking. Milk samples (100 ml) were taken from every gland of each cow

at -24 h, 0 h, +18 h relative to treatment with saline solution and CNH,

where 0 h refer to day of infusion. Milk samples on day 0 were taken

prior to the infusion. At day 0, a dose of 10 mL of CNH [17], with a

6

peptide concentration of 7 mg mL− 1

was infused into each treated

gland with a special applicator following careful sterile cleaning of the

teat. The control glands of the cows in the first group were infused with

10 ml of sterile saline. Milk yield was discarded for 3 days following the

infusion.

Analytical procedures: One set of samples (10 ml) was send to central

laboratory for the determination of total protein, fat, lactose and somatic

cell count [8]. A second set of milk samples (2 x 10 ml) were defatted

under cold conditions [26] and analyzed as follow according to

previously described procedures: concentration of lactose, protein, fat,

casein, whey protein, proteose peptones, lactoferrin, albumin, Na+ and K

+

and the activity of xanthine oxidase, lactoperoxidase, and the

concentration of nitrite (by the DAN reagent), nitrate (by the Griess

reaction), and uric acid [11]. A sub-set of skim milk was ultracentrifuged

and clear milk serum (whey) devoid of membranous particles and casein

micelle pellet were separated [26].

A third set of samples (70 ml) was used to isolate somatic cells from

milk. The samples were centrifuged at 2,000 × g for 30 min at 4°C;

then the fatty fraction and supernatant were removed. Cells from the

bottom layer were suspended in 500 μl of PBS (pH 7.4) containing

0.02% NaN3 and centrifuged twice (400 × g for 15 min at 4 C0) to

concentrate cells. A cell concentration of at least 1 × 107 cells/ml was

obtained, which was measured using Fossomatic 90 (Foss Electric,

Hillerød, Denmark). After separation the cells were lysed by at least 3

freeze-thaw cycles.

The activities of PL, PG, PA, t-PA and u-PA were determined in the

whey and in the re-dissolved casein micelle pellets and isolated somatic

7

cells. Whey, casein micelle pellets (1 mg/ml), cell lysates and other

reagents were dissolved and diluted in 0.05 modified Tris buffer (MTB)

composed of 0.05 M Tris, 0.1 M NaCl, 0.01% twin 80, pH 7.6. The actual

amount of proteins in the reaction mixtures was determined by the

Bradford method.

Plasmin and PG activities of the isolated fractions were measured by

chromogenic assays [27] in a plate reader (Bio Kinetics Reader EL

340; Bio-Tek Instruments) in triplicate, with minor modifications in the

volumes and concentrations used. For PL determination, sample (25 µL)

was added to 225 µL MTB that contained 1.6 mM SpecPL and allowed to

react for 1 h at 37°C in water bath and then read at 405 nm. For PG

determination, sample (100 µL) was added to 100 µL MTB that

contained 3.2 mM SpecPL and 100 µL MTB that contained human u-PA

(280 IU/mL), and the reaction mixture was allowed to react for 1 h at

37°Cin a water bath and then read at 405 nm. Proper blank and control

mixtures were prepared.

Plasminogen activators and their subtypes were determined by

chromogenic reactions as follow: The t-PA activity was measured

according to published procedure [27] by incubating 25 µL samples

dissolved in MTB with 225 µL of 60 mM Tris-HCl, pH 8·5, 0·09%

Tween 20, with 0·375 mg/ml N-methylsulphonyl-D-Phe-Gly-Arg-4-

nitroanilide acetate substrate (Chromozym t-PA). The absorbance at 405

nm was measured for 60 min and the generation of nitroaniline was

determined by the rate of change in absorbance.

The types of PA present in the CN micelles were further

established as follow: (i) Plasminogen activator activity was determined

reaction system that was composed of 25 µL samples nixed with 225 µL

8

MTB that contained bovine PG (32 µg/ml), 1.6 mM SpecPL, with and

without fibrinogen fractions (FIBGN; 16.2 µg/ml), with and without 4

mM of amiloride. Amiloride, was used to inhibit u-PA activity, and the

fibrinogen fractions, to stimulate t-PA activity, which enables to

differentiate between t-PA activity from u-PA [14]. Tissue-PA was

scored as the activity obtained in the presence of fibrinogen fragments

and amiloride, subtracting the activity obtained in the presence of

amiloride, but in the absence of fibrinogen fragments. Plasminogen

activator activity caused by u-PA was scored as the activity obtained in

the absence of fibrinogen fragments, but in the presence of anti-human t-

PA IgG, subtracting the activity obtained in the presence of amiloride.

(ii) Polyclonal rabbit anti-human t-PA IgG was included to a final

concentration of 0.1 mg/ml in the reaction mixture for direct

determination of t-PA activity, and (iii) The effects of PAI-1 at final

concentration of 500 ng/ml was determined in the CN micelles under the

condition of the direct t-PA analysis [28].

Experiment 2. Milk (∼50 ml per cow) was obtained from udders of six

Israeli-Holstein cows producing ~ 40l/day. The sample from each cow

was taken from a mixed yield of a bacterial-free (see experiment 1) single

udder. and was designated as mature milk (MM). After that milking, the

sampled glands in these cows were completely emptied by hand milking.

When no more milk could be obtained by hand milking, the cows were

injected intramuscularly with a dose of 20 international units of oxytocin

(Vetimex, Bladel, Holland). After 3–5 min the MG was hand milked

again, to ensure that any residual milk left in the alveolus was evacuated.

The cows were injected intramuscularly with a dose of 30 international

units of oxytocin and after 3–5 min 30–50 ml of milk were sampled from

9

each of the previously sampled glands; this milk was designated as fresh

milk (FM). The MM and FM samples were stored in dry ice immediately

after sampling, and were transported to a nearby laboratory, where they

arrived at a temperature of 6–10 °C, and were analyzed within less than

20 min for the content of xanthine + hypoxanthine as described before

[29]. The milk defined as FM was fractionated to obtained whey, CN

micelle and somatic cells and these fractions were analyzed for the

content of t-PA, u-PA, PG and PL as described for experiment 1.

Statistical analysis. The results of experiment 1 were analyzed by using

repeated-measures analysis to model correlated residuals within cow as

described previously [10]. The analysis concentrated on the effects of

treatment, day, and treatment × day interactions. The effects of parity

and of days in milk were not significant (P > 0.25) and therefore were not

included in the analyses presented here.

Results

Treating MG with CNH induced dramatic changes in the secretion

and composition of the treated glands, whereas no significant changes

were recorded in the treated gland (Table 1). Milk yield fell 5.5 folds in

comparison to pre-treated or control levels, and lactose concentration

drooped 7.7 folds, so that lactose secretion was reduced by ~42 folds.

Na+ concentration increased ~ 4 folds whereas K

+ concentration

decreased ~ 4 folds, so that their level in milk of treated glands resembled

the expected level of Na+ and K

+ in blood plasma. Protein concentration

increased in the skim milk of treated glands by ~22%, which could be

related to increase of 78% in whey protein concentration. This was

particularly associated with dramatic increase in the concentration of

proteose peptones (CN degradation peptides), and soluble components of

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the acquired (IgG,), and innate (lactoferrin, albumin, lactoperoxidase,

xanthine oxidase) and related metabolites (uric acid, nitrite and nitrate).

CNH treatment was also associated by ~ 14 folds increase in the treated

glands in the count somatic cells, which are composed mainly of

leukocytes.

Most of PA activity in pre-treated and control glands was

associated with the CN micelle and only minority was found in milk

serum and somatic cells (Table 2). CNH treatment induced increase in PA

activity in CN micelle and somatic cells. However, the activity of PA in

CN micelle per ml milk was 4 folds higher than in somatic cells. No PG

activity could be detected in milk serum or in the somatic cells (data not

shown in a table).

t-PA activity in CN micelle in pre-treated, control and treated gland

accounted for the vast majority of PA activity (Table 3). CNH treatment

induced dramatic reduction in PG activity without change in total PG +

PL activity. Thus, the large increase in PL activity in the CNH treated

glands can be related to conversion of PG to PL and was associated with

dramatic reduction in PG to PL ratio (Table 3).

PA activity was measured with the following additions in CN

micelles and somatic cells (Table 4): Amiloride and anti-u-PA antibody

did not affect PA activity in CN micelles, but reduced it in somatic cells.

PAI-1 and anti t-PA antibody dramatically reduced PA activity in CN

micelles, but not in somatic cells. Addition of fibrin increased PA activity

in CN micelles, but not on somatic cells.

The concentration of xanthine + hypoxanthine in milk defined as

freshly secreted was 38.5 ± 5 µM. On the other hand, no xanthine +

hypoxanthine could be detected in milk defined as mature milk, or in

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freshly secreted milk stored at 37 0C for 30 min (data not shown in a

table). Based on that, we can conclude that freshly secreted milk

represent milk that was sampled within 10 min or less after being secreted

[29]. The content of t-PA, PG and PL in freshly secreted milk was found

to be similar to those found in the pre-treated and control glands (Table

4).

Discussion

It is well established that amiloride affect u-PA but not t-PA, that

PA-1 affect t-PA but not u-PA and that fibrin accelerate t-PA activity but

not u-PA activity [12-14 23, 28]. Based on that, it can be concluded that

under physiological conditions, t-PA in milk is vastly associated with the

CN micelle, whereas, no u-PA cannot be traced in the micelle. This

conclusion is further supported by the interaction with respective anti-

bodies to t-PA and u-PA. As mentioned, this conclusion is also consistent

with some previous reports [6,12, 22]. However, what’s unique to this

study is that the great increase in t-PA activity was induced by CNH. In

previous studies, we show that CNH induce accelerated MG involution,

which is associated with intense activation of the MG immune system [9-

11]. This conclusion is supported by the data presented in Table 1, which

demonstrate that the treatment with CNH precipitously reduced

mammary secretion, disrupts the tight junction (increase Na+ and

decrease in K+ concentrations), induce degradation of CNH, and

activates various elements of the innate and acquired immune system.

These aspects were considered in detail in previous publications [4, 9-11]

and therefore will not consider here in detail. However, our data is

consistent with the theory that the PL system plays a key role in inducing

MG involution by degradation of CN micelle and liberating or inducing

the formation active components that in turn affect MG epithelial cells to

commit involution. In this study, we have identified t-PA as the principal

PA, which is responsible for the conversion of PG to PL.

Our results are also consistent with previous ones, which show that

PG is closely associated with the casein micelle [12, 24]. However,

perhaps the most novel finding in this study, namely, the presences of t-

PA and PG in the CN micelles of freshly secreted milk provide a new

12

insight into the setting of PL-casein interaction. It was already

demonstrated that t-PA is produced and secreted by MG cells [12, 30].

Thus, the close presence of t-PA and CN micelles should not be

surprising as they both share the same excretory pathway through

secretory vesicles released from the Golgi apparatus and because t-PA

has high affinity to the CN micelles [16, 23]. Following early suggestion

[16], PG is generally considered to leak to milk from blood plasma.

Though, to the best of our knowledge; there is no evidence that support

this notion. The presence of PG in fresh milk strongly suggest that it is

secreted into milk through the secretory vesicles route embedded within

the CN micelles along with its activator, t-PA and based on the results

Sorrel et al [28] , most likely along with PA-1 because it secreted by MG

cells [30].

The close association between PL, t-PA, PA-1 and CN suggests

that these components serve as functional complex that regulate the

liberation of active components from the CN micelle. Such an

arrangement allows effective fine tuning of the CN micelles degradation

process: i. it allow the complex to function as time machine, milk stasis

will result in longest exposure and thus higher degradation, and ii. It

allows fast responsive reaction to relevant systemic hormonal effects,

which either attenuate or stimulate casein hydrolysis [4].

The localization of u-PA with somatic cells and lack of u-PA

activity in milk serum is consistent with previous reports [12, 14, 22]. In

more detailed studies, it was demonstrated that milk u-PA is bound

mostly to u-PA receptors on polymorphonuclear cells. Recently, it was

shown that MG cells respond to lipopolysacharide challenge (pro-

inflammatory stress) by increasing the expression of u-PA [31]. The

physiological role of u-PA was attributed to its ability to induce basal

membrane degradation and thus help to induce inflow of

polymorphonuclear to injure of infected tissue [31].

According to present findings, the reported correlation between

increased u-PA activity and CN hydrolysis during mastitis [24], merely

reflect the fact that they response similarly to the inflammatory stress.

Our results in consistent with those of others [12-14, 22], clearly

indicated that there is no direct relation between u-PA activity and CN

hydrolysis in raw milk during inflammation. The substantial evidence for

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major role of u-PA in casein degradation in stored milk may be explained

by the dissociation of u-PA from its receptor and the tendency of u-PA to

form interaction with CN. There are many reports that show that heat

treatment, such as that applied during pasteurization, inactivate PAI-1 and

increase the association between u-PA and CN [23].

14

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Table 1. Effect of treatment with CNH on milk yield (on a single gland level) and skim milk

composition of protein, casein degradation products (proteose peptpnes), Na, K and

components of the immune system in pre-treated, control and treated glands (mean ± SD)

Measures Pre-treated* Control* Treated*

Milk yield, l/day 8.5 ± 1.5a 8.2 ± 1.4 a 1.5 ± 0.9 b

Lactose, mM 148 ± 5.5 a 142± 6.2 a 19 2. ± 3 b.5

Na+, mM 26.2 ± 4.4 a 29.1 ± 4.6 a 110.0 ± 8.5 b

K+, mM 37.2 ± 5.1 a 33.1 ± 4.8 a 8.2 ± 1.9 b

Total protein, mg/ml 32.4 ± 2.5 a 32.8 ± 2.6 a 39.8 ± 3.1 b

Casein, mg/ml 24.6 ±2.7 a 24.9 ± 2.8 a 25.8 ± 3.4 b

Whey protein, mg/ml 7.8 ± 1.9 a 7. 9 ± 2.0 a 14.0± 2.2 b

Proteose peptones, µg/ml 465 ± 29.5 a 472± 31.2 a 1037 ± 37.5 b

IgG, µg/ml 200.5 ± 19.9 a 307.9 ±25.2 a 998.5 ± 31.3 b

Lactoferrin, µg/ml 175.2 ± 19.1 a 200.1 ± 23.8 a 1425 ± 41.3 b

albumin, µg/ml 127.0 ± 17.9 a 255.6± 18.5 a 600.5 ± 24.9 b

Uric acid, µM 34.7 ± 5.9 a 35.2 ± 6.6 a 75.2 ± 8.7 b

nitrite, µM 0.6 ± 0.2 a 0.8 ± 0.3 a 8.1± 1.1 b

nitrate, µM 25.1 ± 4.7 a 27.1 ± 5.1 a 145.6 ± 9.9 b

Lactoperoxidase, unit/ml 2.5 ± 0.5 a 3.1 ± 1.1 a 14.7± 1.3 b

Xanthine oxidase, unit/ml 11.6 ± 2.9 a 12.6 ± 3.3 a 70.9 ± 5.7 b

Somatic cell count. Number/ml

75000 ± 8200 a 82000 ± 9300 a 1050000 ± 12100 b

*Results from experiment 1.

a, bValues mark by different superscript letter are significantly different, P <0.01 or

lower.

21

Table 2. Effect of CNH treatments on the distribution of PA activity (unit/ml*) in milk

fractions (mean ± SD).

Glands Pre-treated** Control** Treated**

Plasminogen activator (PA)

Casein micelle 115.1 ± 5.8 d 118.0 ±6.6 d 463.2 ± 11.7 e

Milk serum 5.5 ± 2.2a 4.5± 2.5 a 11.2 ± 3.7 b

Milk somatic cells b.d 35.5 ± 5. c 101.1 ± 9.9 d

ml* - Using dilution and protein content activity of PA was calculated to be based on

ml of reconstituted raw milk

** results from experiment 1

a,b,c,d,e Values marked by different superscript letter are significantly differebt, P <

0.01 or lower.

21

Table 3. Effect of CNH treatments on the activities of the PA-PLG-PL (unit/ml*) in isolated

casein micelles in pre-treated, control, freshly secreted milk and treated gland (mean ± SD).

Pre-treated** Freshly secreted***

Control** Treated**

PA unit/ml 115.1 ± 5.8 a 114.7 ± 5.9 a 118.0 ±6.6 a 463.2 ± 11.7 b

t-PA 109.5 ± 5.5 a 110.0 ± 5.7 a 110.5 ± 6.3 a 461. 2 ± 10.7 b

plasminogen 37.5 ± 3.8 a 38.1 ± 4.9 a 40.4 ± 4.3 a 6.1± 3.8 b

Plasmin 5.1 ± 1.9 a 5.2 ± 2.8 a 5.3 ± 3.8 a 42.4 ± 5.2 b

Plasminogen +plasmin

42.6 ± 6.1 a 43.1 ± 6.8 a 42.7 ± 5.8 a 48.5 ± 7.3 a

Plasminogen/plasmin ratio

8.4 ± 1.1 a 7.3 ± 1.5 a 8.6 ± 2.1 a 1.1 ± 0.5 b

ml* - Using dilution and protein content activities of PA, PLG and PL were calculated

to be based on ml of reconstituted raw milk

** results from experiment 1

*** results from experiment 2

a,b,c,d,e Values marked by different superscript letter are significantly differebt, P <

0.01 or lower.

22

Table 4. Effect of amiloride, anti-U-PA, PA-I and fibrin on PA activity (unit/ml*) in isolated

casein micelles and isolated somatic cells (mean ± SD).

treatments Casein micelle somatic cells

Pre-treated** 120.2 ± 5.8 a 10.5 ± 1.1 a

Amiloride 118 ± 6.1 a. 1.9 ± 0.9b

Anti u-PA 115 ± 7.1 a 0.9± 1.1b

Anti t-PA 15.7 ± 4.9b 10.4 ± 1.3 a

Fibrin 185 ± 7.9b 10.5 ± 1.5 a

PA-I 25.0 ± 5.5b 10.3 ± 1.4 a

ml* - Using dilution and protein content activity of PA was calculated to be based on

ml of reconstituted raw milk

Milk from pre-treated gland (6 cows) was used to isolate casen micelle and somatic

cells.

a,b Values mark by different superscript letters are significantly different from the

pre-treated values by pair-t-test analysis ; P <0.01 or lower.