Indian Journal of Experimental Biology Vol. 40, April 2002, pp. 477-485
Design and synthesis of hormonotoxin for selective targeting of gonadal cells
Anis Alam, Ranjit C Singh & Vinod Singh*
*Hormone Biochemistry Laboratory, Institute of Self-Organising Systems and Biophysics, and Department of Biochemistry (AA), North-Eastern Hill University, Permanent Campus, Shillong 793 022, India
With the aim of developing a cytotoxic agent that could selectively target the appropriate steroid producing cells in the gonads, hormone-toxin conjugates were synthesized with the usc of different heterobifunctional cross linking agents (HBCLA) such as, 2-iminothiolane HCI (2IT), N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP, 8.6 A) and N- succinimidyl 6-[3-(2-pyridyldithio) propionamido] hexanoate (LC-SPDP, 15.6 A). A complete physico-chemical, immuno- chemical and biochemical analysis was performed. The linkage occurred through the £-NH2 groups of a-subunit of oLH as analysed by RP-HPLC analysis. But it was difficult to ascertain the site of linkage in hCG-gelonin conjugate. A I: I (hor- mone:gelonin) molar ratio was obtained when determined with spectrophotometric, gel electrophoresis and amino acid composition methods. The competitive displacement analysis indicates that the binding occurs via the hormone part and leaving gelonin free which was probed with the gelonin antibodies. The conjugates exhibited comparable immunoreactivity but the receptor binding and cytotoxicity of the conjugates prepared with 21T were higher than the hormonotoxin prepared with the usc of SPDP or LC-SPDP. Therefore, it is concluded that higher receptor binding and cytotoxicity may be due to the retention of positive charge on the lysine residues of oLH, which was preserved during the conjugation process. How- ever, the cytotoxicity of oLH based hormonotoxins remained unaffected with the use of long chain spacer arm that is be- lieved to be used generally to avoid steric hindrance. Based on the data presented here, it may be concluded that it is possi- ble to synthesize oLH or hCG based hormonotoxins which specifically interacted with LH/hCG receptor and significantly inhibited protein synthesis in the tumor cells bearing receptors for gonadotropins. The detailed in vivo experiments under in- vestigation would further demonstrate their effectiveness. If successful, a new class of luteolytic may be available in future.
The selective targeting of tumor cells using specific artificial hybrid proteins possessing both the functions of cell membrane specific recognition and cytotoxic- ity has been under investigation during last several yearsl.7. Such cell type artificial hybrid molecules were synthesized by covalent cross-linking to mono- clonal antibodies specific to the tumors cell to the highly cytotoxic molecules and these hybrid are called
*Correspondent author:
Tel: +91-364-550-028 Fax: +91-364-550-076
Email: vinodsingh56@yahoo.co.in Abbreviations:
21T , 2-iminothiolane. DTT , Dithiothreitol. FSH, Follicle stimu- lating hormone, HBCLA, Heterobifunctional cross-linking agent.
hCG, Human corionic gonadotropin. LC-SPDP, N-succinimidyl- 6-[3-(2-pyridyldithio) propionamido]hexanoate. LH, Luteinizing hormone. oLH , Ovine luteinizing hormone. oLH-SPDP, SPDP modified derivative of oLH. RIP, Ribosome inactivating protein.
RP-HPLC, Reverse-phase high performance liquid chromatogra- phy. SDS-PAGE, Sodium dodecyl sulphate-polyacrylamide gel electrophoresis. SPDP, N-sllccinimidyl-(2-pyridyldithio) propio- nate
as "immunotoxin"t.7. Such molecules, after binding to the target antigens are endocytosed and the toxin is realesed which irreversibly inactivates the protein synthesis machinery of the cells. These conjugates have raised the promise of effective immunotherapy for tumors and graft-versus-host disease. Immunotox- ins have particular potential in the treatment of lukeimias and lymphomas8.9
, although some evidence suggests that they are effective against solid tu-
10 II S I I " .
mors ' . evera extreme y actIve IInmunotoXIl1S have been constructed by covalently linking mono- clonal antibodies to bacterial toxins such as Pseudo- monas exotoxin or to plant toxin such as the ricin A chain, saporin and geloninI2.t5 . The plant RIPs which catalytically inactivate 60S ribosomal unit have been used in the the design of immunotoxinI6.19
. Like anti- bodies, hormones are quite specific in their interaction with receptor. Analogous to immunotoxins, we con- structed "hormonotoxins" (so called because the hor- mone component of the construct provides specificity for toxic action) by linking gonadotropic hormones to
478 INDIAN J EXP BIOL, APRIL 2002
the plant ribosome inactivating protein, gelonin to target hybrid complex to selected cells in the go-
d ~O-2-1 S . I . .
na s- . orne superactlve 10rmonotoxln usmg chemically truncated diphtheria toxin have also been reported24. The present paper describes the design, synthesis of bioeffective hormonotoxins based on ovine luteinizing hormone (oLH) and human chori- onic gonadotropin (hCG) hormones for selective tar- geting to the gonadal cells.
Design of Hormonotoxin
The pituitary and placental glycoprotein hormones of which LH, FSH and hCG are a part constitute a family with unique structural features. They are all similar hormones with distinct functions. LH and hCG are similar both structurally and functionally.
These hormones consists of two non-covalently linked subunits called as a and ~ subunit, both of which are glycosylated25.26. The perfect union of the two subunits is absolutely essential for the effective interaction with the receptor and expression of full biological activit/ 5.26. The perfect union of the two subunits is absolutely essential for the effective inter- action with the receptor and expression of full bio- I og. lca actiI .V. ity 25 26 ' . Th e comp I ete ammo aC. . I d se- quence of the sub units and the arrangement of the carbohydrate units are known. While the subunit within the species are identical, the ~-subunit is des- ignated as hormone specific because its interaction with a-subunit determines hormonal specificit/ 5.26. Thus, the a-subunit in these can be interchanged giv- ing rise to a complex which has hormonal activity, dictated by the choice of the ~_subunit25.26.
Extensive investigations have been carried out to understand the role of different amino acid side chains in the a and ~ subunits in the assembly of hormone structure and biological action, viz. receptor binding, cyclase activation and intracellular response27.28
. All the studies point out the critical importance of the full integrity of the a-subunit for complete biological ac- tivity. The common a-subunit is most sensitive to chemical modifications and thus is important for the biological activity. In contrast, the hormone specific
~-subunit, particularly that of LH and hCG can toler- ate many alterations in the polypeptide moiety with- out seriously compromising biological functions27
.28.
Thus, the amino groups of oLH/hCG ~-subunit can be modified to produce a derivative whose ability to re- combine with an intact a-subunit is not reduced and
subsequently the complex will interact effectively with the receptor.
In one study related to the glycoprotein hormones,
~hCG was linked to ricin-A chain via a disulfide bond2o.21
. The conjugate was toxic to R2C cells hav- ing receptors for hCG. However, as compared to na- ti ve ricin, this ~-hCG-ricin hybrid had very low toxic- ity even to the rat leydig R2C cells2
!. The above stud- ies reveal the critical role of the a-subunit in the bind- ing of hCG (and other glycoprotein hormones) to the receptors. Thus it is not surprising that the omission of the a-subunit from the recombinant was responsi- ble for the low inhibitory activity of the conjugates. Therefore, we initiated systematic studies (as de- scribed below) in order to design and synthesise bioeffective hormonotoxin to selectively target toxic proteins to the specific cells in the gonads22.29-36. Al- though, hCG was used to stud/3 but due to its limita- tion and more availability of oLB, oLH has been ex- tensively used22.29-36.
The most common strategy to link two proteins of interest (in case of immunotoxins) without depleting the biological activity of the reacting protein is via the creation of new -S-S- bridge through their accessible amino groups on the lysine residues. This was usually carried out by using the reagent called as SPDP. Since the ~-subunit could withstand the chemical modifica- tion, an attempt was made to synthesise the hor- monotoxin in which RIP was linked to a_oLH34. Al- though, the SPDP modified ~-oLH can recombine to native a-subunit but ~-oLH-S-S-gelonin conjugate failed to recombine to native a_oLH34.37. Therefore, native oLH was used to covalently link to gelonin. A single E-NH2 group modified by SPDP showed re- duced receptor binding and immunoreacti vity but steroidogenic activity was relatively unaltered32.38. A I: I oLH-S-S-gelonin retained substantial receptor binding and significant cytotoxicity to leydig tumor cells which possess receptors for gonadotropin22.29.36.
A physico-chemical analysis of these conjugates re- veal that the coupling occurs via the a_subunit22.29-36.
Based on this observation, an attempt wa also made to design a "Universal" hormonotoxin. The idea is based on the fact that within species, the glycoprotein hormones, such as LH, TSH, FSH and placental go- nadotropins have common a-subunit while the hor- mone specific ~-subunit can be interchanged to pro- duce a recombinant with the specificity dictated by the choice of ~-subunit25-28. Therefore, the a-subunit
ALAM el al.: DESIGN & SYNTHESIS OF HORMONOTOXIN 479
was conjugated to gelonin and the purified cxoLH-S- S-gelonin was then allowed to recombine to native ~
oLH. Interestingly, the conjugate could not recom- bine, therefore the bioeffective hybrid complex could not be generated37
. In a pre-recombination strategy, the cx-oLH-SPDP recombine the native ~oLH and the complex regained biological activity. However, when the complex was allowed to react to the gelonin-SH, the ~-subunit dissociated from the complex and cxoLH-S-S-gelonin could only be obtained which ob- viously did not possess bioeffectivit/ s.
As, the amino groups of alpha-subunit of gonado- tropins (especially LH and hCG) are critical for recep- tor binding2s.27, an approach in which this part of the hormone is left unmodified was chosen. It was sug-
d . I' d' ?7?9 h II ' f
geste In ear ler stu les- '- t at a amll10 groups 0 beta LH are accessible and could tolerate neutral or positive charge substitution but not introduction of negative charge, SPDP modified beta-oLH recom- bines with free alpha-oLH and regains full biological activity31 . But disulfide linked beta-oLH-gelonin (beta-oLH-S-S-gelonin) failed to reassociate with free alpha-oLH31. Based on these observations, native hormones were used to prepare defined hormonotox- ins and several batches were prepared by using differ- ent cross-linking methods. These well defined and extensively characterized hormonotoxins are de- scribed below.
Materials and Methods
Materials used and the methods of experimenta- tion, such as conjugation of gonadotropins to gelonin by the heterobifunctional cross linking agents such as SPDP, LC-SPDP or 2IT, radioiodination, immunore- activity determination, radioreceptor assay, reverse- phase high performance liquid chromatography (RP- HPLC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), binding and cyto- toxicity assay were described in several publica- tions22.29-37.
Synthesis of hormonotoxin
Prior to conjugation, a study on modification of c- amino group(s) of oLH32-3S.39-41 gelonin42-46 and hCG33 with different heterobifunctional cross linking agents (HBCLA) on immunoreactivity and bioactivity was carried out to determine molar ratio of hor- mone:HBCLA or gelonin:HBCLA for the preparation of hormonotoxin. The details of these modification studies are presented in the accompanying paper47. A careful titration of amino group(s) modification in
oLH/hCG and gelonin revealed that 1:2 molar ratio of protein versus gelonin resulted in thiolation of 1.2+0.2 amino group(s) per molecule. For conjugation, HBCLA modified gelonin, treated with dithiothreitol (DIT) (to generate gelonin-SH) was mixed with HBLCA modified gonadotropin hormones. This con- jugation mixture was subjected to gel-filtration chro- matography on appropriated columns. The ascending portion of the protein peak eluting before oLH/hCG (assumed as hormone-toxin conjugate) was subjected to extensive characterization29-31.33.36.37.
Characterization of hormonotoxin
The conjugates purified by gel-filtration chroma- tography was subjected to RP-HPLC chromatograms, it was evident that amino group modification primar- ily occurred in alpha-oLH and therefore, conjugation
d d . h' b ' ?9-31333637 Th . LH procee e via t IS SU Ul1lt- . . . . us, 111 0 -
gelonin hormonotoxin, the beta-oLH remained uncon- jugated and was dissociated during RP-HPLC analy- sis29-31.33,36,37. However, for hCG-gelonin conjugates, it was difficult to define the site of coupling due to in- complete separation of alpha and beta subunits under the conditions used in RP-HPLC analysis33. The chemical structure of the hormonotoxins synthesized and characterized are recorded in Table 1.
The molar ratio of oLH/hCG and gelonin in the conjugates was determined by different methods.
Spectrophotometrically, the molar concentration of pyridine-2-thione liberated after mixing gelonin-SH to oLH/hCG-HBCLA, was estimated using molar ex- tinction coefficient reported earlier48
. Amino acid analysis and SDS-PAGE49
(under reduced condition) provided new tools to determine molar ratio in hor- monotoxins29-31.33,36,37. The molar ratio of hormone and gelonin in the conjugates as determined by these methods are recorded in Table 2. These data clearly reveal that the conjugation procedure was reproduci- ble. Further studies on immunoreactivity, receptor binding and steroidogenic activity as described below also supported the present conclusion.
The immunoreactivity of the conjugate was ana- lysed by specific radioimmunoassay of oLH/hCG and gelonin and the values are recorded in Table 2.
Thiolation of oLH and hCG reduced immunoreactiv- ity up to 90% and 80% respectively which was not much altered after conjugation. Similar results were also obtained for gelonin reactivity. When corrected for increase in molecular mass of the complex, the overall immunoreactivity (compared to geloninl hormone-HBCLA) was found to be unaffected (as shown in the parentheses of column B of Table 3).
480
Conjugate code
CI
CII
CIIl
C41A
C42A
C43A
AOOla
AOOlb
AOOlc
AOOld
AOOle
C200ARA
C39ARA
C210ARA
C220ARA
C230ARA
Spacer arm (A)
13.6
13.6
13.6
13.6
13.6
13.6
13.6
13.6
13.6
13.6
13.6
13.6
13.6
22.4
22.4
31.2
INDIAN J EXP BlaL, APRIL 2002
Table I-Structure of the conjugates Cross-linking agent used
Activation of Activation of homone with gelonin with
Structure
oLH-S-S-gclonin
H a a H
SPDP SPDP I II II I .
oLH-N-C-(CH2)2-S-S-(CH2)2--C-N-gelonln
H a a H
SPDP SPDP I II II I .
oLH- N-C-(CH2l2-S-S-(CH2)2-C-N-gelonln
H a 0 H
SPDP SPDP I II II I .
oLH-N-C-(CH2)2-S-S-(CH2)2-C-N--gelonln
H
a
0 HSPDP SPDP I II II I .
oLH-N-C-(CH2)2-S-S-(CH2)2-C-N-gelonln
H
a
0 HSPDP SPDP I II II I .
oLH-N-C-(CH2)2- S- S-(CH2l2-C-N-gelonln
H a a H
SPDP SPDP I II II I .
oLH-N-C-(CH2l2-S-S-(CH2)2-C-N-gelonln
H
a a
HSPDP SPDP I II II I .
oLH-N-C-(CH2)2-S-S-(CH2)2-C-N--gelonln
H a a H
SPDP SPDP I II II I .
oLH-N- C-(CH2)2-S-S-(CH2)2-C-N---gelonln
H a () H
SPDP SPDP I II II I .
oLH-N- C- (CH2l2-S-S-(CH2l2-C-N-ge:onln
SPDP SPDP
H
a
Q HI II 11 I , .
oLH-N-C-(CH2l2-S-S-(CH2b-C-N-ge,onln
H a 0 H
SPDP SPDP I II II I .
oLH-N-C-(CH2l2-S-S-(CH2l2-C-N--gelonlrl
H a 0 H
SPDP SPDP I II II I .
oLH-N-C-(CH2l2-S-S-(CH2l2-C-N--gelonln
H
a
0 HSPDP SPDP I II II I .
oLH-N-C-(CH2l2-S-S-(CH2l2-C-N-gelonln
SPDP LC-SPDP H 0 Q H ~
T
I II II I - -{;- N - gebnin
hCG-N-C-(~)2-S-S-(CH2)2C-N - ('::His
LC-SPDP SPDP
a H
H a H 0
II
II
II I II S-S-'CH,j -C-N -geloninhCG-N-C-(CHis-N-G-(CHi2- \ .. 2
LC-SPDP LC-SPDP o HO ~~ ~ 1
.
HI II I II ·CH )-C-N-gelonln
-.N-G-(CH )-S-S-(CH ) -C-N-l 2 s hCG-N-C -(CH2
,s
22 2(COI1Id)
Conjugate code
C150-A2
C160-A2
CI70-A2
C40A
Conjugates
CI50-A2*
CI60A-02*
CI70A-02*
C200ARA**
C2IOARA**
C220ARA**
C230ARA**
AOOla#
AOOlb#
AOOlc#
AOOld#
AOOle#
C39ARA##
C4IA##
C42A##
C43A##
C40A##
Spacer arm (A)
13.6
ALAM el af.: DESIGN & SYNTHESIS OF HORMONOTOXIN
Table I-(Contd) Cross-linking agent used
Activation of Activation of homone with gelonin with
oLH+ -S-S-geIonin
2IT SPDP
2IT SPDP
2IT SPDP
hCG-S-S-gelonin
SPDP SPDP
Structure
Table 2-Molar ratio of oLH and gelonin in the conjugates as determined by different method Conjugate code Molar ratio (oLH : gelonin)
Spectrophotometric Gel-electrophoresis Amino acid Immuno-reactivity
composition
oLH+ -S-S-gelonin
I: 1.2 1:1 1:1
1:1 I: 1.1 1:1
1:1 1:1 1:1
oLH -S-S-gelonin
1:1.2 I: 1.1 1:1
I: 1.1 I: 1.2 1:1
1:1 I: 1.2 1:1
1:1 I: 1.2 1:1
1.8: 1.0 2.7: 1.0
1.0: 1.0 1.25: 1.0
1.0: 1.0 1.0: 1.0
1.0: 1.8 1.0:2.0
1.0:2.0 1.0:2.78
1.0: 1.2 1.0:1.35 1.0: 1.0
1.0: 1.0 1.0:2.0 1.0: 1.0
1.0: 1.3 1.0:2.0 1.0: 1.0
hCG-S-S-gelonin
1.0: 1.0 1.0: 1.0 1.0: 1.0
481
"These values were obtained from ref.37. **These values were obtained from ref.36. #These values were obtained from ref. 50. ##These values were obtained from ref.33.
482 INDIAN J EXP BIOL, APRIL 2002
Table 3 - Immunoreactivity of hormone and Gelonin in the conjugates
Hormone/gelonin/ Hormone assay Gclonin assay
oLH-S-S-gelonin %Cross - reactivit:[ % Cross-reactivity
conjugates A B A B
oLH+ -S-S-gelonin
CI50A-A2* 12.6 44.15 11.90 45.2
CI60A-A2* 11.90 41.50 12.20 46.30
CI70A-A2* 13.00 45.40 12.60 47.98
oLH-S-S-gelonin
C200ARA** 4.80 33.20 17.50 45.80
C210ARA** 4.20 29.00 16.50 54.54
C220ARA** 3.50 31.87 16.00 48.48
C230ARA** 3.20 29.06 16.00 53.60
C39ARA#
C4IA# 4.80 47.20 11.60 48.33
C42A# 5.20 51.30 12.40 51.66
C43A# 5.60 55.06 9.95 41.45
AOOla## 8.60 65.13 9.10 24.30
AOOlb## 7.20 54.50 16.20 43.24
AOOlc## 6.60 49.94 18.10 48.33
AOOld## 4.30 32.54 21.20 69.86
AOOle## 3.60 27.25 28.40 75.82
hCG-S-S-gelonin
C40A# 4.80 47.20 11.60 48.33
Valucs in column A and B indicate the immunoreactivity with respect to native hormone or gclonin and modificd hor- mone or gelonin respectively. *Thcse valucs wcre obtaincd from reL37 .. *'These valucs werc obtaincd from ref.36.
#Thcsc valucs wcre obtained from rer.33. ##Thcsc values were obtained from reL50.
The receptor binding ability was determined by ra- dio-receptor assay employing rat testicular homoge- nate as a source of gonadotropin receptor an steroi- dogenic activity was determined by granulose cell assa/9.31.33.3(,.37. On one hand, modification of a single amino group resulted in drastic loss of receptor bind- ing activity, on the other hand, surprisingly the steroi- dogenic ability was enhanced (Table 4). The receptor binding was reduced to 2-2.5 times after conjugation to gelonin (when gelonin-HBCLA molecular mass was corrected). When calculated by taking oLH- HBCLA or hCG-HBCLA as 100%, the similar pattern was also obtained. Steroidogenic activity was drasti- cally hampered upon conjugation of oLH-HBCLA or hCG-HBCLA to gelonin (Table 4).
Cytotoxicity of Hormonotoxins to Leydig Tumor Cells
The hormonotoxin binds to leydig tumor cells as shown indirectly by employing specific antibodies to gelonin and radioiodinated protein-A as described several publications29.31.33.36.37. When hormonotoxins
were incubated in the presence of native gonadotropin hormones (oLH/hCG), the binding was significantly inhibited which suggested th t the binding of hor- monotoxin leydig tumor cells takes place via hor- mone2.-3t.33.36.37.50. The cytotoxicity in terms of inhibi- tion of protein synthesis was performed as described earl ier29.31.33.36.37 .50.
Hormonotoxins showed concentration dependent inhibition of protein synthesis (up to 90%) showing their in vivo efficacy. The tumor cells devoid of go- nadotropin receptors (such as SP2/0) did neither show binding nor cytotoxicity when incubated for 24 hr.
The Leydig tumor cells pretreated with honnonotox- ins when inoculated (sc) in C57BLl6 mice, the rate of tumor development was significantly inhibited. This also indirectly demonstrate the bioeffectiveness of the hormonotoxins.
The major aim of the present investigation was to develop a cytotoxic drug that could selectively target the appropriate steroid producing cells in the gonads. This is based on the fact that the gonadal cells are the predominant cells which express the gonadotropin
ALAM el al.: DESIGN & SYNTHESIS OF HORMONOTOXIN 483
Table 4 -Receptor binding activity, Ribosome-inactivating property and steroidogenic activity of conjugates Hormone/gelonin/ Receptor binding activity Protei n-synthesis-i nhi bitor acti vi t y Steroidogenic
oLH-S-S-gelonin % reactivit:t % reactivit:t activity (%)
conjugate A B A B
oLH+-gelonin
CI50A-A2* 2.60 25.50 12.30
CI60A-A2* 2.30 22.50 11.80
CI70A-A2* 3.30 32.30 12.90
oLH-gelonin
C200ARA** 1.60 18.85 5.20 34.80 3.20
C210ARA** 1.56 18.36 4.40 36.50 3.60
C220ARA** 1.20 17.20 5.00 33.40 2.60
C230ARA** 1.50 21.45 4.00 33.30 2.50
AOOla## 1.60 100 22.00
AOOlb## 1.20 75.01 18.50
AOOlc## 0.94 58.75 14.20
AOOld## 0.91 56.88 9.60
AOOle## 0.72 45.00 6.30
C39ARA# 0.75 9.37 3.20
C4IA# 1.80 19.78 5.6
C42A# 1.87 20.55 6.20
C43A# 1.60 17.58 5.6
hCG-gelonin
C40A 0.833 8.66 6.30
Values in column A and B indicate the receptor binding and protein synthesis inhibition activity with respect to native hormone or gelonin and modified hormone or gelonin respectively.
*These values were obtained from ref.37. **These values were obtained from ref.36. #These values were ob- tained from ref.33. ##These values were obtained from ref.50.
receptors and thus interact with gonadotropic hor- mone. The action of these hormones is mediated by interaction with high affinity and specific receptors.
The idea is to use the receptors on the gonadal cell surface as homing device to attract cytotoxic hybrid agents carried by LH/hCG for the circulation. Once, the hormone-toxin complex reaches the gonadal cells, the toxin component would be released whereupon protein-synthesis would be terminated resulting in complete cessation of cell function. Without adequate steroid production, reproducti ve events are disrupted. The LH and hCG were used as carrier due to the pres- ence of their high affinity receptors on gonadal cells and their action is mediated by internalization. Based on the data presented here, it may be concluded that it is possible to synthesize oLH or hCG based hor- monotoxins which specifically interacted with LH/hCG receptor and significantly inhibited protein synthesis in the tumor cells bearing receptors for go- nadotropins. The detailed in vivo experiments under investigation would further demonstrate their effec-
tiveness. If successful, a new class of luteolytic may be available in future. The superactive honnonotoxins prepared by covalently linking of gonadotropin to truncated diphtheria was found to be 100 times active than of oLH-gelonin hormonotoxin24. It would of in- terest to compare the toxicity of both the hormonotox- ins in the common tumor cell line for comparative data.
Acknowledgement
This investigation was supported by a Fellowship and a grant from the Rockefeller Foundation, ew York. The financial support from the UGC and CSlR, New Delhi is acknowledged. Authors thank Prof.
M. R. Sairam, Clinical Research Institute of Montreal, Montreal, Canada and Prof. Roy Curtiss III, Chair- man, Biology Department, Washington University, St.
Louis, Mo, for their keen interest and support.
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484 INDIAN 1 EXP SIOL, APRIL 2002
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