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Cristina Zennaro, Federica Tonon, Paola Zarattini, Milan Clai, Alessandro Corbelli, Michele Carraro, Marialaura Marchetti, Luca Ronda, Gianluca Paredi, Maria Pia Rastaldi, Riccardo Percudani
The renal phenotype of allopurinol-treated HPRT-deficient mouse
www.plosone.org
www.plosone.org
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2.0183 0 Td
(s.r.l,)Tj
1.8765 0 Td
(San)Tj
1.6781 0 Td
(Pietro)Tj
2.6475 0 Td
(al)Tj
0.9015 0 Td
(Natisone,)Tj
4.0195 0 Td
(Italy\))Tj
2.33 0 Td
(and)Tj
1.7348 0 Td
(water)Tj
2.4321 0 Td
(ad)Tj
1.1792 0 Td
(libitum.)Tj
-27.9607 -1.2983 Td
(Mice)Tj
2.228 0 Td
(were)Tj
2.126 0 Td
(treated)Tj
2.9933 0 Td
(starting)Tj
3.2655 0 Td
(from)Tj
2.2053 0 Td
(embryonic)Tj
4.5411 0 Td
(day)Tj
1.6384 0 Td
(1214)Tj
2.6419 0 Td
(with)Tj
1.9956 0 Td
(allopurinol)Tj
4.6374 0 Td
(\(Sigma-Aldrich)Tj
6.4176 0 Td
(Co.,)Tj
-35.8863 -1.2982 Td
(St)Tj
0.9921 0 Td
(Louis,)Tj
2.6475 0 Td
(MO,)Tj
2.0863 0 Td
(USA\))Tj
2.4548 0 Td
(or)Tj
1.0998 0 Td
(vehicle)Tj
2.982 0 Td
(administered)Tj
5.4936 0 Td
(continuously)Tj
5.4368 0 Td
(in)Tj
1.0318 0 Td
(the)Tj
1.474 0 Td
(drinking)Tj
3.7077 0 Td
(water)Tj
2.4321 0 Td
(to)Tj
1.0261 0 Td
(pregnant)Tj
-32.8646 -1.3039 Td
(females[)Tj
0.83 0.64 0.02 0 k
(6)Tj
0 g
(],)Tj
4.5977 0 Td
(and)Tj
1.7291 0 Td
(was)Tj
1.7008 0 Td
(continued)Tj
4.2916 0 Td
(after)Tj
2.0353 0 Td
(birth)Tj
2.1883 0 Td
(until)Tj
2.109 0 Td
(sacrifice.)Tj
3.702 0 Td
(Allopurinol)Tj
4.8926 0 Td
(was)Tj
1.6951 0 Td
(dissolved)Tj
3.9004 0 Td
(in)Tj
1.0319 0 Td
(1)Tj
0.6973 0 Td
(N)Tj
-34.5711 -1.2983 Td
(NaOH)Tj
2.9026 0 Td
(at)Tj
0.9524 0 Td
(100)Tj
1.6498 0 Td
(mg/ml)Tj
2.897 0 Td
(concentration)Tj
5.8223 0 Td
(and)Tj
1.7291 0 Td
(then)Tj
2.0183 0 Td
(diluted)Tj
3.0387 0 Td
(in)Tj
1.0318 0 Td
(the)Tj
1.474 0 Td
(drinking)Tj
3.7077 0 Td
(water)Tj
2.4321 0 Td
(to)Tj
1.0262 0 Td
(the)Tj
1.4683 0 Td
(final)Tj
2.0069 0 Td
(concen-)Tj
-34.1572 -1.2983 Td
(tration)Tj
2.9423 0 Td
(of)Tj
1.0148 0 Td
(75)Tj
/F13 1 Tf
1.1735 0 Td
[()]TJ
/F8 1 Tf
(g/ml.)Tj
-3.9344 -1.3039 Td
(Mice)Tj
2.228 0 Td
(were)Tj
2.126 0 Td
(sacrificed)Tj
4.0024 0 Td
(at)Tj
0.9525 0 Td
(1)Tj
0.6973 0 Td
(or)Tj
1.0998 0 Td
(2)Tj
0.6917 0 Td
(months)Tj
3.2825 0 Td
(of)Tj
1.0148 0 Td
(age.)Tj
1.7688 0 Td
(Urine)Tj
2.5682 0 Td
(samples)Tj
3.3845 0 Td
(were)Tj
2.1203 0 Td
(collected)Tj
3.7417 0 Td
(from)Tj
2.2054 0 Td
(the)Tj
1.4683 0 Td
(bladder)Tj
-34.5484 -1.2982 Td
(and)Tj
1.7291 0 Td
(examined)Tj
4.1272 0 Td
(by)Tj
1.1849 0 Td
(phase)Tj
2.4831 0 Td
(contrast)Tj
3.4582 0 Td
(and/or)Tj
2.9424 0 Td
(stereo)Tj
2.6022 0 Td
(microscopy)Tj
4.8755 0 Td
(\(Leica)Tj
2.6419 0 Td
(Microsystems,)Tj
5.9867 0 Td
(Wetzlar,)Tj
3.6 0 Td
(Ger-)Tj
-35.6312 -1.2983 Td
(many\).)Tj
3.0387 0 Td
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2.2904 0 Td
(blood)Tj
2.5114 0 Td
(collection,)Tj
4.337 0 Td
(serum)Tj
2.7213 0 Td
(was)Tj
1.7007 0 Td
(separated)Tj
4.0082 0 Td
(and)Tj
1.7348 0 Td
(stored)Tj
2.7042 0 Td
(at)Tj
0.9525 0 Td
(-80C.)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 419.4708 cm
BT
/F0 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Blood)Tj
2.7401 0 Td
(values)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 402.4629 cm
BT
/F8 1 Tf
1 TL
-0.004 Tc
0 0 Td
(Blood)Tj
2.5851 0 Td
(urea)Tj
1.9786 0 Td
(nitrogen)Tj
3.651 0 Td
(\(BUN\))Tj
2.948 0 Td
(and)Tj
1.7348 0 Td
(xanthine)Tj
3.7304 0 Td
(oxidase)Tj
3.2031 0 Td
(activity)Tj
3.1181 0 Td
(were)Tj
2.126 0 Td
(determined)Tj
4.8472 0 Td
(using)Tj
2.3924 0 Td
(commercial)Tj
-32.3147 -1.2982 Td
(kits)Tj
1.644 0 Td
(according)Tj
4.1839 0 Td
(to)Tj
1.0262 0 Td
(the)Tj
1.4683 0 Td
(manufacturer)Tj
5.6919 0 Td
(indications)Tj
4.6658 0 Td
(\(Sigma-Aldrich)Tj
6.4233 0 Td
(Co.\).)Tj
2.177 0 Td
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2.8403 0 Td
(creatinine)Tj
4.2236 0 Td
(was)Tj
-34.3443 -1.3039 Td
(measured)Tj
4.1045 0 Td
(by)Tj
1.1849 0 Td
(HPLC)Tj
2.7496 0 Td
(method.)Tj
3.5489 0 Td
(Briefly)Tj
2.8516 0 Td
(12.5)Tj
/F13 1 Tf
1.8709 0 Td
[()]TJ
/F8 1 Tf
(l)Tj
0.9694 0 Td
(of)Tj
1.0148 0 Td
(sera)Tj
1.8142 0 Td
(were)Tj
2.1203 0 Td
(deproteinized)Tj
5.7373 0 Td
(by)Tj
1.1792 0 Td
(adding)Tj
2.9934 0 Td
(1)Tj
0.6916 0 Td
(ml)Tj
1.2869 0 Td
(of)Tj
1.0148 0 Td
(100%)Tj
-35.1323 -1.2983 Td
(acetonitrile.)Tj
4.9549 0 Td
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3.4922 0 Td
(were)Tj
2.126 0 Td
(then)Tj
2.0183 0 Td
(vortexed)Tj
3.6907 0 Td
(and)Tj
1.7291 0 Td
(centrifuged)Tj
4.7848 0 Td
(twice)Tj
2.3188 0 Td
(for)Tj
1.3833 0 Td
(20)Tj
1.1735 0 Td
(minutes)Tj
3.4639 0 Td
(at)Tj
0.9524 0 Td
(4C,)Tj
1.9786 0 Td
(16000)Tj
2.5965 0 Td
(g.)Tj
-36.663 -1.2983 Td
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2.2847 0 Td
(vacuum)Tj
3.4072 0 Td
(evaporation)Tj
4.9833 0 Td
(of)Tj
1.0148 0 Td
(the)Tj
1.4683 0 Td
(supernatant)Tj
4.9833 0 Td
(fraction,)Tj
3.5829 0 Td
(dried)Tj
2.3358 0 Td
(samples)Tj
3.3845 0 Td
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(suspended)Tj
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(in)Tj
-34.0041 -1.2982 Td
(12.5)Tj
/F13 1 Tf
1.8708 0 Td
[()]TJ
/F8 1 Tf
(l)Tj
0.9751 0 Td
(of)Tj
1.0148 0 Td
(5)Tj
0.6917 0 Td
(mM)Tj
1.9332 0 Td
(NaH)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 284.2582 335.7921 cm
BT
/F8 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(2)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 287.8299 337.4929 cm
BT
/F8 1 Tf
1 TL
-0.004 Tc
0 0 Td
(PO)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 300.8692 335.7921 cm
BT
/F8 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(4)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 304.4409 337.4929 cm
BT
/F8 1 Tf
1 TL
-0.004 Tc
0 0 Td
(,)Tj
0.4422 0 Td
(pH)Tj
1.508 0 Td
(6.4)Tj
1.3946 0 Td
(\(mobile)Tj
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(phase\))Tj
2.8233 0 Td
(and)Tj
1.7291 0 Td
(stored)Tj
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(at)Tj
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(-20C.)Tj
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(manually)Tj
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3.3902 0 Td
(by)Tj
1.1792 0 Td
(a)Tj
0.652 0 Td
(10)Tj
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1.1735 0 Td
[()]TJ
/F8 1 Tf
(l)Tj
0.9751 0 Td
(loop)Tj
1.9956 0 Td
(on)Tj
1.2755 0 Td
(a)Tj
0.6519 0 Td
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(HPLC)Tj
2.7496 0 Td
(system)Tj
2.9367 0 Td
(\(Shimadzu,)Tj
4.7792 0 Td
(Kyoto,)Tj
2.8856 0 Td
(Japan\).)Tj
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(a)Tj
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(SCX)Tj
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(column)Tj
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(\(Zorbax)Tj
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(SCX-300)Tj
3.7928 0 Td
(4.6)Tj
1.3946 0 Td
(x)Tj
0.686 0 Td
(250,)Tj
1.8708 0 Td
(Agilent)Tj
3.1521 0 Td
(Technologies,)Tj
5.76 0 Td
(Santa)Tj
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(Clara,)Tj
2.5908 0 Td
(CA,)Tj
1.7972 0 Td
(US\))Tj
1.7688 0 Td
(with)Tj
1.9956 0 Td
(an)Tj
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(isocratic)Tj
3.5716 0 Td
(run)Tj
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(and)Tj
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(revealed)Tj
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(234)Tj
1.6441 0 Td
(nm)Tj
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(and)Tj
1.7348 0 Td
(240)Tj
1.6441 0 Td
(nm.)Tj
1.8142 0 Td
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(were)Tj
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1.7291 0 Td
(analyzed)Tj
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(software.)Tj
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(output)Tj
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1.9956 0 Td
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(creatinine)Tj
4.2236 0 Td
(standards)Tj
4.0818 0 Td
(\(Sigma-Aldrich\).)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 242.9858 cm
BT
/F0 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Purine)Tj
3.0708 0 Td
(HPLC)Tj
2.8631 0 Td
(analysis)Tj
3.7842 0 Td
(of)Tj
1.0441 0 Td
(mouse)Tj
3.189 0 Td
(sera)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 225.9779 cm
BT
/F8 1 Tf
1 TL
-0.004 Tc
0 0 Td
(Mouse)Tj
2.931 0 Td
(sera)Tj
1.8084 0 Td
(obtained)Tj
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(centrifugation)Tj
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(of)Tj
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(blood)Tj
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(-80C)Tj
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(until)Tj
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7.0468 0 Td
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(performed)Tj
4.4674 0 Td
(by)Tj
1.1792 0 Td
(adding)Tj
2.9934 0 Td
(0.4)Tj
1.3946 0 Td
(M)Tj
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(perchloric)Tj
4.3029 0 Td
(acid)Tj
1.8709 0 Td
(at)Tj
0.9524 0 Td
(the)Tj
1.4684 0 Td
(ratio)Tj
2.0976 0 Td
(1:1.67)Tj
2.5738 0 Td
(\(v/v\))Tj
2.1373 0 Td
([)Tj
0.83 0.64 0.02 0 k
(11)Tj
0 g
(].)Tj
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(samples)Tj
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(overnight)Tj
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1.7178 0 Td
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(20)Tj
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(4C,)Tj
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(16000)Tj
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(new)Tj
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(tubes,)Tj
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(and)Tj
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(at)Tj
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(-20C.)Tj
2.8006 0 Td
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(\(10)Tj
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1.5137 0 Td
[()]TJ
/F8 1 Tf
(l\))Tj
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(HPLC)Tj
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(system)Tj
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(Shimadzu,)Tj
4.4391 0 Td
(Kyoto,)Tj
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(C18)Tj
1.8369 0 Td
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(C18)Tj
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(150x4.6)Tj
3.2881 0 Td
(mm,)Tj
2.0863 0 Td
(Agilent)Tj
3.1521 0 Td
(Technologies,)Tj
5.7543 0 Td
(Santa)Tj
2.4094 0 Td
(Clara,)Tj
2.5966 0 Td
(CA,)Tj
1.7914 0 Td
(US\))Tj
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(and)Tj
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(254)Tj
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(and)Tj
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(270)Tj
1.6497 0 Td
(nm.)Tj
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7.353 0 Td
(runs)Tj
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(dual)Tj
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(phases)Tj
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(gradient)Tj
3.5547 0 Td
([)Tj
0.83 0.64 0.02 0 k
(11)Tj
0 g
(].)Tj
2.0749 0 Td
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1.7858 0 Td
(mobile)Tj
-33.9531 -1.2983 Td
(phase)Tj
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(A)Tj
0.9014 0 Td
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4.2009 0 Td
(0.5)Tj
1.3947 0 Td
(mM)Tj
1.9332 0 Td
(sodium)Tj
3.2314 0 Td
(pentanesulfonate)Tj
7.0469 0 Td
(and)Tj
1.7292 0 Td
(10)Tj
1.1735 0 Td
(mM)Tj
1.9332 0 Td
(KH)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 474.6897 120.359 cm
BT
/F8 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(2)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 478.2614 122.0031 cm
BT
/F8 1 Tf
1 TL
-0.004 Tc
0 0 Td
(PO)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 491.3007 120.359 cm
BT
/F8 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(4)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 494.8724 122.0031 cm
BT
/F8 1 Tf
1 TL
-0.004 Tc
0 0 Td
(,)Tj
0.4422 0 Td
(pH)Tj
1.508 0 Td
(3.5)Tj
1.3946 0 Td
(and)Tj
1.7291 0 Td
(the)Tj
-34.5596 -1.2982 Td
(mobile)Tj
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(phase)Tj
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(B)Tj
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(has)Tj
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(the)Tj
1.474 0 Td
(same)Tj
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(composition)Tj
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(of)Tj
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(phase)Tj
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(A)Tj
0.9015 0 Td
(in)Tj
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(10%)Tj
1.9276 0 Td
(acetonitrile)Tj
4.7281 0 Td
(\(v/v\).)Tj
2.364 0 Td
(The)Tj
1.7858 0 Td
(output)Tj
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(peaks)Tj
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(where)Tj
2.6532 0 Td
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(and)Tj
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0.9524 0 Td
(48)Tj
1.1679 0 Td
(and)Tj
1.7348 0 Td
(96)Tj
1.1678 0 Td
(hours)Tj
2.5172 0 Td
(after)Tj
2.0352 0 Td
(xanthine)Tj
3.7303 0 Td
(or)Tj
1.0998 0 Td
(uric)Tj
1.8085 0 Td
(acid)Tj
1.8709 0 Td
(treatments.)Tj
-30.8123 -1.2982 Td
(MTT)Tj
2.3357 0 Td
(was)Tj
1.6951 0 Td
(given)Tj
2.3811 0 Td
(to)Tj
1.0261 0 Td
(cells)Tj
1.9219 0 Td
(at)Tj
0.9525 0 Td
(the)Tj
1.4683 0 Td
(final)Tj
2.0012 0 Td
(concentration)Tj
5.8224 0 Td
(of)Tj
1.0148 0 Td
(0.5)Tj
1.3946 0 Td
(mg/ml.)Tj
3.1237 0 Td
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2.2903 0 Td
(4)Tj
0.6917 0 Td
(hours)Tj
2.5171 0 Td
(of)Tj
1.0148 0 Td
(incubation)Tj
4.5468 0 Td
(at)Tj
-36.1981 -1.2983 Td
(37C,)Tj
2.4491 0 Td
(cell)Tj
1.559 0 Td
(medium)Tj
3.6057 0 Td
(was)Tj
1.7008 0 Td
(removed)Tj
3.7474 0 Td
(and)Tj
1.7291 0 Td
(the)Tj
1.4683 0 Td
(salt)Tj
1.5647 0 Td
(crystals)Tj
3.1691 0 Td
(formed)Tj
3.1577 0 Td
(inside)Tj
2.6135 0 Td
(the)Tj
1.4684 0 Td
(cells)Tj
1.9219 0 Td
(were)Tj
2.1203 0 Td
(solubilized)Tj
-32.275 -1.3039 Td
(with)Tj
1.9956 0 Td
(dimethyl)Tj
3.787 0 Td
(sulfoxide)Tj
3.8324 0 Td
(\(DMSO\).)Tj
3.9742 0 Td
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4.9889 0 Td
(was)Tj
1.7008 0 Td
(then)Tj
2.0183 0 Td
(read)Tj
1.9728 0 Td
(at)Tj
0.9524 0 Td
(570)Tj
1.6498 0 Td
(nm)Tj
1.5873 0 Td
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2.3868 0 Td
(a)Tj
0.6576 0 Td
(spectropho-)Tj
-31.5039 -1.2983 Td
(tometer)Tj
3.3619 0 Td
(\(Spectra)Tj
3.5036 0 Td
(Max)Tj
2.0125 0 Td
(Plus)Tj
1.9106 0 Td
(384,)Tj
1.8708 0 Td
(Molecular)Tj
4.2973 0 Td
(Devices,)Tj
3.532 0 Td
(Sunnyvale,)Tj
4.558 0 Td
(CA,)Tj
1.7914 0 Td
(USA\).)Tj
2.6816 0 Td
(Cellular)Tj
3.3789 0 Td
(cytotoxicity)Tj
-32.8986 -1.2982 Td
(was)Tj
1.6951 0 Td
(evaluated)Tj
3.9911 0 Td
(by)Tj
1.1849 0 Td
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3.0671 0 Td
(Dehydrogenase)Tj
6.4233 0 Td
(\(LDH\))Tj
2.9423 0 Td
(assay)Tj
2.2677 0 Td
(\(Bio)Tj
1.9104 0 Td
(Vision)Tj
2.8687 0 Td
(Inc.,)Tj
1.9786 0 Td
(Milpitas,)Tj
3.7303 0 Td
(CA\).)Tj
2.1317 0 Td
(MDCK)Tj
-34.1912 -1.304 Td
(cells)Tj
1.9219 0 Td
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2.1203 0 Td
(seeded)Tj
2.9083 0 Td
(as)Tj
1.0148 0 Td
(in)Tj
1.0318 0 Td
(MTT)Tj
2.3357 0 Td
(assay)Tj
2.2677 0 Td
(protocol)Tj
3.5943 0 Td
(and,)Tj
1.9559 0 Td
(at)Tj
0.9525 0 Td
(the)Tj
1.4683 0 Td
(end)Tj
1.7235 0 Td
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1.0147 0 Td
(the)Tj
1.4683 0 Td
(appropriate)Tj
4.8756 0 Td
(treatment,)Tj
4.3653 0 Td
(100)Tj
/F13 1 Tf
1.6441 0 Td
[()]TJ
/F8 1 Tf
(l)Tj
-36.663 -1.2982 Td
(of)Tj
1.0148 0 Td
(cellular)Tj
3.1351 0 Td
(supernatant)Tj
4.9833 0 Td
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2.1259 0 Td
(transferred)Tj
4.6375 0 Td
(to)Tj
1.0261 0 Td
(corresponding)Tj
6.0604 0 Td
(wells)Tj
2.1769 0 Td
(in)Tj
1.0375 0 Td
(an)Tj
1.2019 0 Td
(optical)Tj
2.914 0 Td
(clear)Tj
2.1203 0 Td
(96-well)Tj
-32.4337 -1.2983 Td
(plate.)Tj
2.3641 0 Td
(100)Tj
/F13 1 Tf
1.644 0 Td
[()]TJ
/F8 1 Tf
(l)Tj
0.9752 0 Td
(of)Tj
1.0148 0 Td
(reaction)Tj
3.4922 0 Td
(solution,)Tj
3.7077 0 Td
(consisting)Tj
4.269 0 Td
(of)Tj
1.0148 0 Td
(catalyst)Tj
3.1691 0 Td
(solution)Tj
3.4808 0 Td
(and)Tj
1.7348 0 Td
(dye)Tj
1.6214 0 Td
(solution)Tj
3.4866 0 Td
(in)Tj
1.0318 0 Td
(a)Tj
0.6576 0 Td
(ratio)Tj
-33.6639 -1.2983 Td
(1:45,)Tj
2.0919 0 Td
(were)Tj
2.1203 0 Td
(then)Tj
2.0183 0 Td
(added)Tj
2.6589 0 Td
(to)Tj
1.0261 0 Td
(each)Tj
2.0239 0 Td
(well)Tj
1.8199 0 Td
(and)Tj
1.7291 0 Td
(incubated)Tj
4.1726 0 Td
(for)Tj
1.3833 0 Td
(30)Tj
1.1735 0 Td
(minutes)Tj
3.4638 0 Td
(at)Tj
0.9468 0 Td
(room)Tj
2.4264 0 Td
(temperature,)Tj
5.3518 0 Td
(protect-)Tj
-34.4066 -1.3039 Td
(ing)Tj
1.5023 0 Td
(the)Tj
1.4684 0 Td
(plate)Tj
2.1373 0 Td
(from)Tj
2.2053 0 Td
(light.)Tj
2.2507 0 Td
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4.989 0 Td
(was)Tj
1.6951 0 Td
(finally)Tj
2.7042 0 Td
(measured)Tj
4.1102 0 Td
(at)Tj
0.9524 0 Td
(500)Tj
1.6497 0 Td
(nm)Tj
1.5874 0 Td
(using)Tj
2.3868 0 Td
(a)Tj
0.6576 0 Td
(spectrophotome-)Tj
-30.2964 -1.2982 Td
(ter)Tj
1.3096 0 Td
(\(Spectra)Tj
3.5036 0 Td
(Max)Tj
2.0126 0 Td
(Plus)Tj
1.9162 0 Td
(384,)Tj
1.8708 0 Td
(Molecular)Tj
4.2917 0 Td
(Devices\).)Tj
3.8777 0 Td
(As)Tj
1.2643 0 Td
(positive)Tj
3.3164 0 Td
(control,)Tj
3.3448 0 Td
(Triton)Tj
2.8233 0 Td
(X-100)Tj
2.6533 0 Td
(\(1%)Tj
1.7914 0 Td
(final)Tj
-33.9757 -1.2983 Td
(concentration\))Tj
6.1568 0 Td
(treated)Tj
2.999 0 Td
(cells)Tj
1.9219 0 Td
(were)Tj
2.1203 0 Td
(considered,)Tj
4.8245 0 Td
(whereas)Tj
3.447 0 Td
(free)Tj
1.7234 0 Td
(medium)Tj
3.6056 0 Td
(was)Tj
1.7007 0 Td
(used)Tj
2.058 0 Td
(as)Tj
1.0148 0 Td
(negative)Tj
-31.572 -1.3039 Td
(control.)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 475.37 cm
BT
/F0 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Cell)Tj
1.9134 0 Td
(immunostaining)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 458.3622 cm
BT
/F8 1 Tf
1 TL
-0.004 Tc
0 0 Td
(Cells)Tj
2.1599 0 Td
(were)Tj
2.1203 0 Td
(fixed)Tj
2.194 0 Td
(in)Tj
1.0318 0 Td
(4%)Tj
1.457 0 Td
(paraformaldehyde/0.15%)Tj
10.2671 0 Td
(picric)Tj
2.4888 0 Td
(acid)Tj
1.8708 0 Td
(for)Tj
1.389 0 Td
(20)Tj
1.1679 0 Td
(minutes)Tj
3.4639 0 Td
(at)Tj
0.9524 0 Td
(room)Tj
2.4264 0 Td
(tempera-)Tj
-32.9893 -1.2982 Td
(ture.)Tj
2.0636 0 Td
(Afterwards,)Tj
4.8925 0 Td
(cells)Tj
1.9162 0 Td
(were)Tj
2.126 0 Td
(washed)Tj
3.1748 0 Td
(thrice)Tj
2.5341 0 Td
(with)Tj
1.9956 0 Td
(PBS)Tj
1.8369 0 Td
(and)Tj
1.7291 0 Td
(incubated)Tj
4.1782 0 Td
(with)Tj
1.9956 0 Td
(0.5%)Tj
2.1543 0 Td
(Triton)Tj
2.8233 0 Td
(X-100)Tj
2.6476 0 Td
(for)Tj
-36.0678 -1.2983 Td
(5)Tj
0.6916 0 Td
(minutes,)Tj
3.6964 0 Td
(then)Tj
2.0182 0 Td
(with)Tj
1.9956 0 Td
(6%)Tj
1.4513 0 Td
(FBS/PBS)Tj
3.7474 0 Td
(for)Tj
1.389 0 Td
(1)Tj
0.6916 0 Td
(hour)Tj
2.1543 0 Td
(at)Tj
0.9525 0 Td
(room)Tj
2.4264 0 Td
(temperature)Tj
5.1307 0 Td
(and)Tj
1.7291 0 Td
(finally)Tj
2.7042 0 Td
(exposed)Tj
3.4469 0 Td
(to)Tj
1.0205 0 Td
(the)Tj
-35.2457 -1.2982 Td
(primary)Tj
3.4525 0 Td
(antibody)Tj
3.7644 0 Td
(\(E-cadherin)Tj
5.006 0 Td
(1:3200,)Tj
3.05 0 Td
(Sigma)Tj
2.6816 0 Td
(Aldrich)Tj
3.2655 0 Td
(Co.\))Tj
1.9559 0 Td
(overnight)Tj
4.0818 0 Td
(at)Tj
0.9525 0 Td
(4C)Tj
1.7518 0 Td
(in)Tj
1.0374 0 Td
(a)Tj
0.652 0 Td
(humid)Tj
2.8913 0 Td
(cham-)Tj
-34.5427 -1.304 Td
(ber.)Tj
1.7461 0 Td
(After)Tj
2.2847 0 Td
(three)Tj
2.262 0 Td
(quick)Tj
2.4435 0 Td
(washes,)Tj
3.2371 0 Td
(cells)Tj
1.9219 0 Td
(were)Tj
2.1203 0 Td
(incubated)Tj
4.1782 0 Td
(with)Tj
1.9956 0 Td
(the)Tj
1.474 0 Td
(secondary)Tj
4.269 0 Td
(anti-rat)Tj
3.2201 0 Td
(Alexa)Tj
2.4775 0 Td
(Fluor)Tj
2.3981 0 Td
(488)Tj
-36.0281 -1.2982 Td
(\(Molecular)Tj
4.6317 0 Td
(probes\))Tj
3.2485 0 Td
(diluted)Tj
3.0387 0 Td
(1:400,)Tj
2.5682 0 Td
(for)Tj
1.389 0 Td
(1)Tj
0.6973 0 Td
(hour)Tj
2.1543 0 Td
(at)Tj
0.9524 0 Td
(room)Tj
2.4208 0 Td
(temperature)Tj
5.1307 0 Td
(in)Tj
1.0375 0 Td
(the)Tj
1.4683 0 Td
(dark.)Tj
2.2734 0 Td
(After)Tj
2.2847 0 Td
(washing,)Tj
-33.2955 -1.2983 Td
(slides)Tj
2.4037 0 Td
(were)Tj
2.126 0 Td
(mounted)Tj
3.8721 0 Td
(by)Tj
1.1792 0 Td
(Moviol)Tj
3.0954 0 Td
(Mounting)Tj
4.2746 0 Td
(solution)Tj
3.4809 0 Td
(\(Fluka,)Tj
3.0218 0 Td
(Sigma-Aldrich)Tj
6.0774 0 Td
(Co.\).)Tj
2.1827 0 Td
(Images)Tj
3.0614 0 Td
(were)Tj
-34.7752 -1.3039 Td
(acquired)Tj
3.702 0 Td
(with)Tj
1.9956 0 Td
(a)Tj
0.6519 0 Td
(Leica)Tj
2.3074 0 Td
(DM-2000)Tj
4.1045 0 Td
(microscope.)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 335.6787 cm
BT
/F0 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Statistical)Tj
4.4504 0 Td
(analysis)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 318.6708 cm
BT
/F8 1 Tf
1 TL
-0.004 Tc
0 0 Td
(Data)Tj
2.1203 0 Td
(are)Tj
1.4513 0 Td
(presented)Tj
4.1159 0 Td
(as)Tj
1.0148 0 Td
(mean)Tj
/F13 1 Tf
2.4434 0 Td
[()]TJ
/F8 1 Tf
0.788 0 Td
(SEM)Tj
2.16 0 Td
(or)Tj
1.0999 0 Td
(mean)Tj
/F13 1 Tf
2.4434 0 Td
[()]TJ
/F8 1 Tf
0.788 0 Td
(standard)Tj
3.7247 0 Td
(deviation)Tj
3.9458 0 Td
(and)Tj
1.7292 0 Td
(statistical)Tj
3.9117 0 Td
(significance)Tj
4.938 0 Td
(\(P)Tj
-36.6744 -1.2982 Td
(value\))Tj
2.6475 0 Td
(was)Tj
1.6951 0 Td
(calculated)Tj
4.2066 0 Td
(by)Tj
1.1792 0 Td
(using)Tj
2.3924 0 Td
(Instat)Tj
2.5002 0 Td
(2.0)Tj
1.3946 0 Td
(and)Tj
1.7291 0 Td
(Graphpad)Tj
4.269 0 Td
(6.1)Tj
1.3946 0 Td
(software)Tj
3.5886 0 Td
(applying)Tj
3.6681 0 Td
(ANOVA)Tj
3.7643 0 Td
(and)Tj
1.7348 0 Td
(T-)Tj
-36.1641 -1.2983 Td
(test)Tj
1.5987 0 Td
(as)Tj
1.0148 0 Td
(appropriate.)Tj
5.1023 0 Td
(P)Tj
0.7824 0 Td
(values)Tj
/F17 1 Tf
0 Tc
2.6645 0 Td
(<)Tj
/F8 1 Tf
-0.004 Tc
(0.05)Tj
2.6476 0 Td
(were)Tj
2.1203 0 Td
(considered)Tj
4.6034 0 Td
(to)Tj
1.0205 0 Td
(be)Tj
1.1508 0 Td
(statistically)Tj
4.6091 0 Td
(significant.)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 261.0141 cm
BT
/F2 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Results)Tj
/F0 1 Tf
0 -1.5023 Td
(Survival)Tj
3.7323 0 Td
(and)Tj
1.8614 0 Td
(renal)Tj
2.4095 0 Td
(morphology)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 225.9779 cm
BT
/F8 1 Tf
1 TL
-0.004 Tc
0 0 Td
(Treatment)Tj
4.4446 0 Td
(with)Tj
2.0013 0 Td
(allopurinol)Tj
4.6374 0 Td
(had)Tj
1.7065 0 Td
(highly)Tj
2.7099 0 Td
(toxic)Tj
2.1827 0 Td
(effects)Tj
2.7269 0 Td
(in)Tj
1.0318 0 Td
(HPRT)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 440.0503 230.4566 cm
BT
/F8 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(-/-)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 449.9716 225.9779 cm
BT
/F8 1 Tf
1 TL
-0.004 Tc
0 0 Td
(mice,)Tj
2.3754 0 Td
(as)Tj
1.0148 0 Td
(compared)Tj
4.2462 0 Td
(to)Tj
1.0262 0 Td
(the)Tj
1.4683 0 Td
(same)Tj
-35.1266 -1.2982 Td
(drug)Tj
2.109 0 Td
(dosages)Tj
3.2995 0 Td
(administered)Tj
5.4935 0 Td
(to)Tj
1.0204 0 Td
(HPRT)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 344.8629 217.4173 cm
BT
/F8 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(+/-)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 356.485 212.9952 cm
BT
/F8 1 Tf
1 TL
-0.004 Tc
0 0 Td
(and)Tj
1.7291 0 Td
(wild)Tj
1.9445 0 Td
(type)Tj
1.9162 0 Td
(\(WT\))Tj
2.4775 0 Td
(animals.)Tj
3.5546 0 Td
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1.3947 0 Td
(mice)Tj
2.1543 0 Td
(survived)Tj
3.6056 0 Td
(during)Tj
-34.4236 -1.2982 Td
(the)Tj
1.4683 0 Td
(experimental)Tj
5.4595 0 Td
(procedures.)Tj
4.8983 0 Td
(Nonetheless,)Tj
5.3006 0 Td
(HPRT)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 396.907 204.4346 cm
BT
/F8 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(-/-)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 406.7716 200.0125 cm
BT
/F8 1 Tf
1 TL
-0.004 Tc
0 0 Td
(animals)Tj
3.3335 0 Td
(were)Tj
2.1203 0 Td
(smaller)Tj
3.1237 0 Td
(\()Tj
0.83 0.64 0.02 0 k
(Fig)Tj
1.8199 0 Td
(1a)Tj
1.1281 0 Td
(and)Tj
1.7292 0 Td
(1b)Tj
0 g
(\))Tj
1.542 0 Td
(and)Tj
-35.4724 -1.3039 Td
(feebler)Tj
2.9026 0 Td
(than)Tj
2.0296 0 Td
(HPRT)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 274.9039 191.4519 cm
BT
/F8 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(+/-)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 286.5259 186.9732 cm
BT
/F8 1 Tf
1 TL
-0.004 Tc
0 0 Td
(and)Tj
1.7291 0 Td
(WT)Tj
1.7971 0 Td
(at)Tj
0.9525 0 Td
(age)Tj
1.542 0 Td
(of)Tj
1.0148 0 Td
(1)Tj
0.6973 0 Td
(month)Tj
2.9197 0 Td
(and)Tj
1.7291 0 Td
(were)Tj
2.126 0 Td
(sacrificed.)Tj
-21.9626 -1.2982 Td
(As)Tj
1.2699 0 Td
(compared)Tj
4.2406 0 Td
(to)Tj
1.0261 0 Td
(the)Tj
1.4683 0 Td
(other)Tj
2.347 0 Td
(groups,)Tj
3.1975 0 Td
(allopurinol)Tj
4.6375 0 Td
(treated)Tj
2.999 0 Td
(HPRT)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 449.4047 178.4692 cm
BT
/F8 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(-/-)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 459.3259 173.9905 cm
BT
/F8 1 Tf
1 TL
-0.004 Tc
0 0 Td
(mice)Tj
2.1543 0 Td
(had)Tj
1.7064 0 Td
(increased)Tj
4.0025 0 Td
(kidney)Tj
-33.7943 -1.2982 Td
(weight/body)Tj
5.21 0 Td
(weight)Tj
2.8857 0 Td
(ratio)Tj
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