A (GPA ), (Perco ll) A, Gene Express ion of Erythrocyte M em brane Glycophor in A in N IDDM

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1 14 5 V o l114,n o Ch inese Jou rnal of B iochem istry and M o lecu lar B io logy O cṫ 1998 g GPA 3 (, ) g (N IDDM ) A (GPA ), (Perco ll) N IDDM, RNA, N o rthern GPA GP W estern, N IDDM GPA m RNA ( , P < 0101), 6 N IDDM 3a 3b, 3 3a 3b 4 N IDDM GPA,, N IDDM (RCD ) : ( ), A, Gene Express ion of Erythrocyte M em brane Glycophor in A in N IDDM J i Q iong2m ei 3 L iu Jun2Fan L u Y i2q ing (L aboratory of B lood B iochem istry, H unan M ed ical U niversity, Chang sha ) Abstract To study the gene exp ression of eryth rocyte m em b rane glycopho rin A (GPA ) in patien ts w ith non2in su lin2dependen t diabetes m ellitu s (N IDDM ), hum an reticu locytes w ere separated by discon tinuou s den sity gradien t of perco ll from peripheral b lood of 15 healthy sub jects and 25 N IDDM patien ts, and to tal RNA s of their reticu locytes w ere iso lated U sing the reticu locyte RNA s, the gene exp ression levels of m em b rane GPA w ere estim ated by N o rthern do t hyb ridization W estern b lo tting w as perfo rm ed by alkaline pho sphatase2linked imm unodetection T he con ten ts of GPA m RNA in N IDDM group w ere sign ifican tly increased than tho se of the con tro l group ( vs in tegrated OD, respectively; P < 0101) T he W estern imm unob lo ts of eryth rocyte m em b rane GP revealed ill2defined de2 m arkation betw een bands 3a and 3b in 6 N IDDM patien ts and w eak ly stained bands of 3a, 3b and 4 in o ther 3 patien tṡ T herefo re, it is inferred that the reduction of eryth rocyte m em b rane GPA in N IDDM m ay p rom o te the com pen sato ry elevation of its gene exp ression, and the aberration w ou ld occu r in related tran slation step T h is deduction is po ssib ly in acco rdance w ith the findings of distinct decrease of red cell defo rm ab ility (RCD ) in N IDDM patien tṡ Key words: D iabetes m ellitu s, N on2in su lin2dependen t (N IDDM ), E ryth rocyte m em b rane, Gly2 3 (, ) : , : China Academic Journal Electronic Publishing House All rights reserved

2 copho rin A, Gene exp ression [ 1, 2 N IDDM (RCD ) ], RCD RCD Schw artz [ 3 ] 20 N IDDM, Β2 (Β2sp ec2 trin), 412, 35% 15%,, RCD,,, A (GPA ), [ 4 ] N IDDM, GPA, N o rthern N IDDM GP GPA N IDDM RCD, (1) g W HO (1980) N IDDM 25, 17 8 ; , (2) 15 ( 9, 6), Perco ll2400, (D EPC ), Β2 cdna ( ) ; D T T,N P240,BSA, (Sigm a) ;M O PS (BR I) ; P stg, RN ase, P ro tob lo t (P rom ega) ; (B io2r ad) ; N C (Sch leicher & Schuell) ; Α2 32 P2dCT P ( ) DU 27 (Beckm an), GL 220A ( ), (A pp ligene), 60002IS (Beckm an), (B io2r ad) XL (L KB ) [ 5, ] 017 cm 65%, 60%, 55%, 50% perco ll 013 m l, 015 m l, g 25 m in, ( ), PBS 2, RNA [7 ], g (0114 m o lgl N ac l, 115 mm o lgl M gc l2, 0101 m o lgl T ris2hc l ph 810, 015%N P240, m o lgl D T T ) 200 Λl 012 m o lgl (V RC) 20 Λl,,, 200 Λl g (7 m o lgl, 1% SD S, 0135 m o lgl N ac l, 10 mm o lgl ED 2 TA 2N a2, 10 mm o lgl T ris2hc l, ph 810), g g ( ) 3,, T E, 115 GPA-cD NA M GPA Α(ΑM GPA ) cdna GPA (ph GPA 23) Fukuda, 500 bp cdna, ng cdna, ( Λg DNA cpm ) [8 116 Northern ],, cdna h 3, 30 m in,, XA R 25 (Ko China Academic Journal Electronic Publishing House All rights reserved

3 dak) d XL U ltrascan, [ ],, GP, IgG ( ) (A P), A P, 118, t 2 211, 3,, 212 RNA RNA, 114%, 28S 18S 5S rrna (F ig 1) A 260gA RNA 213 GPA N IDDM RNA, GPA cdna Β2, N o rthern, N IDDM F ig 1 114% agaro se electropho resis of to tal RNA GPA (P hum an reticulocytes (015 TBE, 5 V gcm ) a, b, c, d: H ealthy subjects; e: Size m arkers of mo lecular < 0101), Β2 w eigh t fo r RNA N IDDM ( ), (T ab le 1, F ig 2) Table 1 A comparison of GPA and Β2actin gene exp ression analyzed by N o rthern do t blo tting betw een no rm al subjects and N IDDM patients Gene exp ression Group of no rm al subjects Group of N IDDM patients t test GPA (5 Λg loaded) Β2actin (10Λg loaded) Gases exam ined Integral value of OD (x θ x ) Cases exam ined Integral value of OD (x θ x ) in P < 0101 P > 0105 F ig 2 N o rthern do t blo ts of reticulocyte to tal RNA from two healthy subjects (N ) and three N IDDM patients (D ) hybridized to the GPA cd2 NA p robe 5, 10 and 15 Λg of samp les w ere loaded China Academic Journal Electronic Publishing House All rights reserved

4 214 GP (F ig 3) F ig 3, GPA, GP (a ) [ 9 7 ], 1 (GPA ) 2 (GPA GPB ) 3a 3b (GPB ) 4 (GPA ) 5 (GPD ) 6 (GPB ) 10 N IDDM, 1 (n ) 3b, 9 3a 3b (e m ), 3 4 (k m ) 6 (e j ) 3 (k m ) GP, GP, GPA GPB F ig 3 W estern imm unoblo ts of healthy subjects ( lane a ) and N IDDM patients ( lanes e to n) show ing m em brane GPA and GPB bands 3 RCD,, N IDDM, N a + 2K + 2A T P, [ 10 A T P ], [ 11, ] Schw artz [ 3 ],, N IDDM [ 11 GP ], RCD, [ 4 ], GPA GP, GPA B C D E,, GPA B, M N Ss, [ 4 ], GPA 411 N o rthern, N IDDM GPA 9 GP 3a 3b, 3 GPA ( 4), GPA, RCD, China Academic Journal Electronic Publishing House All rights reserved

5 GPA GPA, GPA 2m RNA RCD References 1 M cm illan D E, Gion K M Gluco sylated hemoglobin and reduced eryth rocyte defo rm ability in diabetes H orm M etab R es, 1981, 11: ,,,,,, g (Song T ian2p ing, Q iu Hong2X in, W ang Shu2P ing, Zhang Su2H ua, Shen P ing2gu, Su Shao2Chu, L i P ing Changes of red blood cell defo rm ability in type g diabetes m ellitus Ch in J E nd ocrinol M etab), 1991, 7: Schw artz R S,M adson J W, Rybick i A O, N agel R L O xidation of spectrin and defo rm bility defects in diabetic eryth ro2 cytes D iabetes, 1991, 40: L u Y Q, L iu J F, H uang C H, B lum enfeld O O, Schw artz R S, L aw rence C, N agel R L E llip tocyto sis associated w ith an abno rm al Αglycopho rin A nn H em atol, 1992, 65: Paw ar V B, H ernick M R,M asters J M A discontinuous density gradient m ethod fo r separating reticulocytes J M ed T echnol, 1984, 1: ,,, ΑgΒ (D ing Chun2J in, Zhu D ing2e r, Yuan T ian2ying T he study of disequilibrium of the bio synthesis of ΑgΒglobin chains in m yelo id leukem ia B u ll H unan M ed U niv), 1993, 18: 5 8 7, DNA RNA (Zhu Xun, Yang Gui2Zhen A simp le m ethod fo r extraction of DNA and RNA from the sam e cell J N orm an B ethune U niv M ed S ci), 1991, 17: Sam brook J,,,, 2, : (Sam brook J, F ritsch EF,M a2 niatis T M olecu lar C loning A L aboratory M anual 2nd ed Co ld Sp ring H arbo r L abo rato ry P ress, 1989 T ranslated by J ing Dong2Yan, L im eng2feng et al, Beijing: Science P ress), 1992: L u Y Q, N icho ls M E, B igbee W L, N agel R L, B lum enfeld O O Structural po lymo rph ism of glycopho rins demonstrated by imm unoblo tting techniques B lood, 1987, 69: ,,, (W ang Chang2 Sen, L u Yi2Q ing, Fu M in2zhuang, L iu Si2Q i Erythrocyte nucleo tide contents in diabetes m ellitus Chin J E ndocrinol M etab), 1989, 5: g (J i Q iong2m ei B iochem ical changes of eryth rocytes in patients w ith N IDDM F oreig n M ed S ci S eries P hy siol P athol S ci & C lin M ed ), 1996, 16: China Academic Journal Electronic Publishing House All rights reserved