Thrombin-mediated phosphoinositide hydrolysis in Chinese hamster ovary cells overexpressing phospholipase C-delta 1.

The regulatory mechanism(s) of a phosphoinositide-specific phospholipase C, PLC-delta 1, was investigated using a clone of stably overexpressed PLC-delta 1 (PLC delta 30 cells) in Chinese hamster ovary cells. Thrombin stimulation of PLC delta 30 cells exhibited 6.5-fold increase in total inositol phosphates (InsP), which was significantly higher than that in the vector-transfected (V1) cells (2.0-fold). AIF-4 increased InsP accumulation in both V1 and PLC delta 30 cells, and pertussis toxin partially blocked InsP accumulation in thrombin-stimulated PLC delta 30 cells. Guanosine thiotriphosphate (GTP gamma S) markedly potentiated thrombin-stimulated InsP generation in permeabilized PLC delta 30 cells compared with V1 cells, suggesting possible involvement of a G-protein (s) in the activation of PLC-delta 1. In PLC delta 30 cells, ionomycin-induced significant InsP generation and thrombin-stimulated InsP generation were completely inhibited by addition of EGTA. Furthermore, the stimulatory effects of thrombin plus GTP gamma S in PLC delta 30 cells were more sensitive to change in free calcium concentration than in V1 cells. Suppression by 12-O-tetradecanoylphorbol 13-acetate of thrombin-stimulated InsP accumulation was not affected by increasing Ca2+ concentration. These results indicate that thrombin-induced PLC-delta 1 activation is regulated via both G-protein(s) and calcium.

amide gel electrophoresis. activation mechanism. When receptor-type tyrosine kinases, such as epidermal growth factor or platelet-derived growth factor receptors are stimulated, tyrosine phosphorylation of PLC--y occurs and induces phosphoinositide breakdown (7)(8)(9). Activation of receptors having no intrinsic kinase also induces phosphorylation of PLC-y via nonreceptor-type tyrosine kinases (2).
Receptors for muscarinic acetylcholine, bradykinin, and thrombin belong to the family of receptors containing seven membrane-spanning domains characteristic of those coupled to effector enzymes through guanine nucleotide-binding protein (G-protein) (10). Recent investigations provided evidences that receptor-mediated activation of PLC-p isozymes is caused by two distinct mechanisms; one through a subunits of Gq family which is insensitive to pertussis toxin (PT) and the other through the Py subunits of G-proteins (11)(12)(13)(14)(15)(16)(17). In vitro reconstitution studies show that activation of the PLC-/3 isozymes mediated by Gqa is in the order of PLC-p1 > PLC-p3 >> PLC-p2, whereas P-y subunits-mediated stimulation of PLC-/3 isozymes is in the order of PLC-p3 > PLC-p2 > PLC-p1 (18,19).
In contrast, despite the wide distribution of PLC-6 isozymes from mammalian cells (20) to yeast (21,22), their receptorlinked activation mechanismb) has not fully been clarified. The PLC-6 isozymes with an approximate molecular mass of 85 kDa are considerably smaller than either PLC-p (130-155 kDa) or PLC-y (145 kDa). PLC-6 isozymes also contain two conservative domains, X and Y, but they lack src homology regions and are therefore unlikely to be a substrate for tyrosine kinases (2). It is also to be noted that PLC-6 isozymes only contain calciumbinding EF-hand motif (23). Therefore, it is conceivable that PLC-6 isozymes are sensitive to calcium and therefore that the intracellular calcium elevation alone may provoke PLC-6 to hydrolyze polyphosphoinositides in vivo. Or the calcium binding to the EF-hand motif may modulate activation process of the PLC-6 such as the translocation to membrane or interaction with putative G-protein(s). Little is known about the type(s) of receptor coupled to PLC-6 except that the PLC-61 is associated with thrombin-triggered mitogenic response (24,25). Two mutants of CCL39 cells lacking PLC-61 showed significant different behaviors in thrombin-induced PI-turnover: decreased and enhanced responses (26). In the present study, in order to get more insight into the regulatory mechanisms of PLC-61, we have investigated the thrombin receptor-mediated activation of phosphoinositide hydrolysis in CHO cells overexpressing PLC-61. (90 CUmmol) was obtained from Amersham (Buckinghamshire, United Kingdom) and [3Hlphosphatidylinosi-to1 4,5-bisphosphate (PIP,, 8.8 CUmmol) was from DuPont NEN. Thrombin was obtained from Mochida Pharmaceutical Company (Tokyo, Japan). Guanosine 5'-O-(y-thiotriphosphate) (GTPyS) and guanosine 5'-O-(P-thiodiphosphate) (GDPpS) were from Boehringer Mannheim. Ionomycin and phorbol myristate acetate (TPA) were purchased from Sigma. PT and digitonin were from Wako (Osaka, Japan). Genistein and Geneticin (G418) were from Funakoshi (Tokyo, Japan) and Life Technologies, Inc., respectively.

EXPERIMENTAL PROCEDURES MateriaZs-my~-[~H]inositol
CrII Cultlrre and ZYansfection of PLC-61 cDNA-CHO cells were maintained in a growth medium of Ham's F-12 supplemented with 10% fetal calf serum. PLC-61 cDNA subcloned in a plasmid vector, pIBI20, was kindly supplied by Dr. S. G. Rhee (National Institutes of Health, Bethesda, MD). The PLC-Gl/pIBIBO was digested with HindIII, and the obtained 2.8-kilobase pair insert was subcloned into a mammalian expression vector, pSRn, containing human immunodeficiency virus promotor and nro gene (27). The constructed plasmid DNA or the vector DNA was transfected into CHO cells seeded on a collagen-coated 60-mm culture dish by the modified CaPO,, method using a mammalian transfection kit (Stratagene, La dolla, CA). Two days after the transfection, the cells were reseeded in a 100-mm culture dish in growth medium containing 0.8 m g h l G418 and further cultured for 3 days. The G418resistant cells were then subcultured in two 96-well microtiter plates to obtain single clones by limiting dilution. Three and 20 clones for vectorand PLC-61-transfected cells, respectively, were obtained and maintained in the presence of 0.2 m g h l G418. The expression of PLC-61 protein was examined by Western blotting with monoclonal anti-PLC-61 antibody using the ECL detection system (Amersham).
M~a.wren7rt1/ of Inositol Phosphates-CHO cells were seeded a t a density of 7 x 10' cells/well in six-well plates in complete growth medium. At near confluence, the growth medium was changed to 1.0 ml of inositol-free minimum Eagle's medium containing 0.3% bovine serum albumin and m,vo-[:'H]inositol (1 uCi/ml). After 36-h labeling, the cells were washed two times with modified Krebs-Ringer buffer (KRB) consistingof 125 m\l NaCI, 5 mil KCI, 1.2 mhi KH,PO,, 1.2 mxl MgSO,, 2 mM CaCI,, 6 my glucose, 25 mxl Hepes (pH 7.4). and 20 my LiCl and were further incubated for 15 min a t 37 "C. Experiments were conducted by adding agonists to the labeled cells, and the reaction was terminated by the addition of 0.5 ml of 10% perchloric acid to each well at the indicated times. Inositol phosphates were separated using Dowex AG1-X8 anion exchange resin (200-400 mesh, formate form, Bin-Rad) a s described elsewhere (281. I:lHIInositol and I:'Hlglycerophosphoinositol were run to waste and the fractions containing InsP,, InsP?, and InsP,, plus InsP,, were collected. CrIl P~rt~~eahilization-l"HIInositol-labeled cells were permeabilized by incubating with 15 p~ digitonin for 6 min a t 25 "C in potassium glutamate (KG) buffer consisting of 139 mv potassium glutamate, 20 mhl Pipes (pH 6.9), 1 mht Mg-ATP, 1 mhl MgCI,, and 5 mv EGTA, containing 20 m\l LiCl a s described previously (29). Under these conditions, more than 98'% of the cells were permeabilized, as assessed by the trypan blue exclusion test. Then, the overlaying medium was aspirated, and the cells were incubated with the indicated concentration of free Ca" or various reagents in potassium glutamate buffer a t 37 "C. The reaction was terminated, and inositol phosphates were determined as described above. Prrparation of C,ytosolic and Men2hmrw Fractions-Confluent cultures of CHO cells in 100-mm dishes were washed three times with 4 ml of ice-cold phosphate-buffered saline and then 2 ml of cold hypotonic buffer (10 mnr Tris/HCI (pH 7.4), 100 mlr NaCI, 1 mv EDTA, 5 mht EGTA, and 1 mli dithiothreitol) containing protease inhibitors (10 pg/ml leupeptin, 0.5 mxl phenylmethylsulfonyl fluoride) were added. The cells were gently scraped from the dish with a rubber policeman, homogenized for 30 strokes with a Dounce homogenizer, After unbroken cells were removed by centrifugation a t 3,000 x g for 5 min, the supernatant fractions were centrifuged a t 105,000 x g for 30 min to give a cytosolic and membrane fractions. The membrane pellet was then resuspended in the hypotonic buffer.
Protein was determined with the Bio-Rad protein assay kit using bovine serum albumin as standard.
Electrophoresis and Immunohlot/ing-SDS-polyacrylamide gel electrophoresis was run through 8 9 acrylamide gel according to the method by Laemmli (31). Electrophoretic blotting onto nitrocellulose membrane (PVDF) was carried out as the procedure of Towbin (32). Blocking was

Overexpression of PLC-61 in CHO Cells-CHO cells were
transfected with a construct containing rat brain PLC-61 cDNA, and three clones (PLC62, -27, -30) were obtained. Aclone PLC630 showing the highest level of PLC-61, as inferred by Western blot analysis, was selected and employed through the following experiments. A clone of vector-transfected CHO cells (V1) was used as a control. No significant differences were observed in growth rate and morphology between the PLC630 and V1 cells (data not shown).
Western blot analyses using antisera against bovine PLC-pl, "yl, and -61 revealed marked overexpression of PLC-61 in the 400 pg in both cell types. I t could be possible that CHO cells also contain other PLC isozyme(s), probably PLC-63, which is widely expressed in a variety of tissues and cell lines, because PLC-p2 is known to be present only in HL-60 (18, 19). Distributions of PLC-y1 and PLC-61 in cytosolic and membrane fractions were examined in CHO cells (Fig. 2). In contrast to PLC-yl which was mainly present in cytosol, a high level of PLC-61 was observed in the membrane fraction of PLC630 cells. When PLC activity was measured with PIP, as substrate, the specific activities of homogenates of V1 and PLC630 cells were 37.9 -t 4.6 and 121.2 -t 1.4 nmol/min/mg of protein, respectively, and the activities of the membrane fractions were 2.5 -t 0.8 and 8. In contrast to the marked responses to thrombin, bradykinin known to cause receptor-mediated phosphoinositide hydrolysis in a variety of cells did not show significant increase in InsP level both in the V1 cells and PLC630 cells (Fig. 5). AIF, increased ["HlInsP production in V1 and PLC630 cells 2.8-fold Ca" dependence of PLC activation by Ca"-mobilizing hormones has been a matter of controversy. For example, hormonestimulated polyphosphoinositide breakdown in intact hepatocytes is independent of the intracellular calcium rise (33, 34), whereas Ca" ionophore A23187 stimulated PLC-mediated breakdown of polyphosphoinositides in rabbit neutrophils (35). This discrepancy may be due to differences of PLC isozymes. Thrombin receptors are known to elevate intracellular Ca2+ levels by stimulating the extracellular Ca" influx (36) PLCS30 cells induced by thrombin was completely blocked by addition of 3 m~ EGTAin the incubation medium (603 2 19.9% for thrombin; 130.2 2 60.4% for thrombin plus EGTA compared with the unstimulated level) (Fig. 5). Artificial rise of intracellular Ca2+ by ionomycin (1 p~, 30 min) in the presence of extracellular Ca2+ (1.5 m~) promoted InsP generation (281.9 * 24.6%) in PLCS30 cells, but it caused only a slight increase of the InsP levels in the control cells (120 2 11.4%). When assayed in the presence of EGTA with no added Ca2+, PLC-61 partially purified from yeast cannot hydrolyze PIP, (21). These results suggest that intracellular Ca2+ is essential for thrombin-stimulated PLC-61 activation in the PLCS30 cells.
Effects of GTPyS and Thrombin in Permeabilized Cells-Thrombin receptor has been known to couple to two G-proteins, Gq and Gi in CCL39 fibroblasts cells (37). In order to obtain further evidence for involvement of G-protein in PLC-61 activation, the effect of GTPyS on InsP formation was examined in digitonin-permeabilized cells of both V1 and PLCS30. The digitonin-permeabilized cells were incubated in the presence or absence of thrombin with increasing concentrations of GTPyS (10-100 p~). As shown in Fig. 6, GTPyS over a range of 10-100 p~ markedly enhanced InsP levels in the presence of thrombin in PLCS30 cells, although GTPyS alone produced only a small increase. In contrast, V1 cells exposed to thrombin in the presence of GTPyS showed merely a very small increase in [3H]InsP level. The effects of GDPpS on the GTPyS-stimulated InsP production were further examined in the both types of cells (Fig. 7). GDPPS (1 m~) inhibited the GTPyS (50 pM)-stimulated [3H]InsP productions in the V1 and PLC630 cells (99.8 2 10.0% and 70.4 9.8% inhibition, respectively), and its partial inhibitions in the V1 and PLC630 cells (65.2 2 10.5% and 51.3 .c 12.5% inhibition, respectively) was observed in the thrombin plus GTPyS-stimulated [3H]InsP production.
In order to see whether the PLC-81 takes a major part in stimulation of phosphoinositide breakdown in PLCS30 cells by thrombin, we examined the effect of anti-PLC-S1 antibody on PIP, breakdown in the membrane fractions prepared from the V1 and PLCS30 cells. The membrane fractions were incubated at 30 "C for 15 min in the presence or absence of thrombin (1 unitlml) plus 100 p~ GTPyS. The PIP, hydrolyzing activities of V1 and PLCS30 cell membranes were stimulated by thrombin plus GTPyS 1. Inhibition oflnositol Phosphate Production by TPA in Thrombin-stimulated Cells-It is well known that the activation of protein kinase C (PKC) exerts a negative feedback to phosphoinositide hydrolysis in most of cell types, as a result of phosphorylation of a constituent(s) in the signaling pathway of the receptor to PLC (3). Recently, it was shown that TPA treatment stimulated in vivo phosphorylation of PLC-p1, but not PLC-61, thereby preventing interaction of PLC-pl with a G-protein (38). To examine whether the TPA affects thrombin-stimulated InsP production in PLC630 cells, the intact cells were pretreated with various concentrations of TPA for 15 min and then stimulated with thrombin. As shown in Fig. 9, TPA caused inhibition of thrombin-induced [3H]InsP production in a similar dose-dependent manner in two cell lines. The TPApretreatment (200 nM for 15 min) caused translocation of cytosolic PKC isozymes (a, 6, E ) to the membrane fraction in CHO cells' (data not shown). To examine if the negative feedback regulation by PKC of phosphoinositide hydrolysis occurs at a post-receptor level, the effects of GTPyS, thrombin, or thrombin plus GTPyS on [3HlInsP formation were investigated in permeabilized PLC630 cells with or without TPA pretreatment. The thrombin-or thrombin plus GTPyS-stimulated [3H]InsP production at 0.1 J~M Ca2+ was suppressed (40% inhibition) by the treatment with TPA (200 nM for 15 min) (Fig. lo), and the suppressive effect by TPA was observed even in the presence of 1 Ca2+. However, TPA did not significantly inhibit GTPyS-induced [3H]InsP production at both low and high Ca2+ concentrations. The suppressive effects by TPA on thrombin-or thrombin plus GTPyS-stimulated InsP production in the V1 cells were almost equivalent to those in PLC630 cells (data not shown). These results suggest that the inhibition of thrombin-stimulated InsP formation by TPA is unlikely to prevent interaction of PLC-61 with a G-protein. DISCUSSION Although the regulatory mechanisms of PLC-p and PLC-y activation have well been studied and characterized, much less is known about the activation mechanism of PLC-6. In order to gain more insight into the mechanism of PLC-61 activation, in the present study, we have investigated its regulatory mechanism using a line of CHO cells transfected with PLC-61 cDNA. It was shown that the production of InsP stimulated with thrombin was markedly enhanced in PLC-61-overexpressed Y. Banno, Y. Okano, and Y. Nozawa, unpublished observation. PLC630 cells compared with vector-transfected control cells, whereas bradykinin (BK) which can stimulate phosphoinositide hydrolysis in a variety of cell types did not produce InsP in these cells. Furthermore, investigations using both intact and permeabilized cells demonstrated that thrombin-induced InsP formation was modulated by the receptor-coupled G-protein(s), the intracellular Ca" concentrations, and the activation of PKC, indicating that the activation mechanisms of PLC-61 were similar but not identical to those of PLC-p type.
Although it is generally known that all PLC isozymes can hydrolyze PIP, dependently on Ca2+ i n vitro, most of the previous studies have shown that hormone-induced phosphoinositide hydrolysis by PLC in intact cells was observed at low intracellular Ca2+ levels of unstimulated cells M) (39). Downes and Michell (40) suggested that agonist-induced hydrolysis of phosphoinositides is relatively insensitive to extracellular Ca2+ removal and that artificial elevation of Ca2+ does not promote the hydrolysis. Our previous data using a cell line of osteoblasts MC3T3-El were consistent with this notion (41). The MC3T3-El cells contain much higher amounts of PLC-p1 and PLC-yl but less PLC-61 relative to PLCS30 cells. It was shown that in MC3T3-El cells BK-stimulated InsP generation was neither affected by extracellular Ca2+ chelation with EGTA of Overexpressed PI-PLC-61 nor intracellular Ca2+ elevation by ionomycin. BK receptors have recently been considered to be coupled to PLC-pl through a family of G-proteins, Gq (13). Thus, BK-induced InsP formation is probably mediated by the activation of PLC-p, and an increase in [Ca2+]i is not required for its activation. However, the present investigation indicated the absolute requirement of extracellular Ca", because its depletion caused a nearly complete inhibition of thrombin-induced InsP production in PLCS30 cells. Furthermore, Ca2+ ionophore ionomycin alone induced greater increases in InsP levels in PLC630 cells than in vector-transfected (Vl) cells which contain PLC-p type and much less PLC-61. In permeabilized PLC630 cells, the [CaZ+l, level up to 1 VI was sufficient to cause a small but significant InsP production, whereas no significant InsP production was observed at the same Ca2+ concentration in V1 cells. These results suggest a preferential association of Ca2+ with PLC-6 compared with PLC-p in activation in vivo. Eberhard and Holz (42) have proposed a hypothetical view that the initial transient cytosolic Ca2+ rise induced by InsP, resulting from receptor/G-protein-mediated PLC(A) activation may in turn contribute to its prolonged activation of PLC(B). Although the identity of these two types of PLC has not yet been defined, it was thought that PLC(A) would be the PLC-p type and PLC(B) may be the same or a different enzyme. Our results obtained with thrombin-stimulated InsP formation in the PLC630 cells appear to be compatible with this view. Thrombin-stimulated phosphoinositide breakdown in PLC630 cells was markedly enhanced relative to V1 cells when incubated for 30 min, but its difference was much smaller at early stimulation time between these two cell lines, suggesting that the activation of PLC-61 is caused a t a later step in thrombin-stimulation. Furthermore, there are some differences between these two cells in response to calcium and PT. Calcium dependence of thrombin-stimulated InsP generation was much higher in PLC630 cells than in the V1 cells. F' T sensitivity for inhibition of the thrombinstimulated InsP generation in PLC630 cells was much higher than that of the V1 cells, suggesting that different types of G-proteins might be involved in these cells. PLC-61, distinct from PLC-/3 and PLC-y, is very unique in that it contains a Ca2+-binding EF-hand motif in its amino acid sequence (23). It is thus conceivable that binding of Ca2' to the EF-hand domain may modulate the substrate selectivity and catalytic activity, or any change in other properties of PLC-61, such as subcellular distribution or interaction with other regulatory proteins, e.g. G-protein(s). The results obtained from the present study using GTPyS indicate that intracellular Ca2+and G-protein act synergistically for PLC-61 activation by thrombin in PLC630 cells.
Recent investigations have shown that PLC-p isozymes are stimulated with G-proteins, and distinct isozymes are stimulated even with distinct subunits of G-proteins; PLC-p1 and PLC-p3 are activated by a-subunits of Gq family and PLC-p2 and PLC-p3 are by py subunits of various G-proteins (11)(12)(13)(14)(15)(16)(17). However, the type of G-protein for activation of PLC-6 isozymes remains to be defined and characterized. We have demonstrated here that GTPyS potentiated the thrombin effect in phosphoinositide breakdown in permeabilized PLC630 cells, suggesting evidence for involvement of putative G-protein.
More recently, Park et al. (18) have demonstrated that PLC-61 as well as PLC-/3 types were activated by G-protein Py subunits in vitro. A distinction between PLC-p1 and PLC-61 is that the Y region in the putative catalytic domain is followed by 450 carboxyl-terminal residues in PLC-p1 but by only 10 residues in PLC-61 (2). It has been demonstrated that the region required for activation by Gaq is localized to the carboxyl-terminal-most region (903-1142) of PLC-p1 (18, 43). On the other hand, P y subunits of G-protein activate PLC-p isozymes by interacting with amino-terminal region but not carboxyl-termi-nal region of PLC-p (44, 45). Thus it is tempting to speculate that GPy subunits could bind to the amino-terminal region of PLC-61. CHO cells contain several receptor-coupled G-proteins (Gi2, Gi3, Gs, and Gq) (46,47). Pretreatment with PT of PLCS30 cells partially inhibited InsP production induced by thrombin, indicating partial involvement of PT-sensitive G-protein(s). Thrombin receptor is known to couple to two G-proteins, Gq and Gi2 (37). Inhibition by PT of thrombin-stimulated InsP formation was much higher in PLC630 cells relative to that of V1 cells which may couple to Gq, suggesting that a different type of G-protein may be involved in PLC-61 activation by thrombin. Stimulation of InsP formation by thrombin plus GTPyS was also observed in the membrane from PLC630 cells, but the stimulation level was much less compared with those obtained in whole cell or permeabilized cells.
The PLC-y isozyme has SH domains which are required for their activation (7-91, but the PLC-6 isozyme has no such domains, indicating that tyrosine phosphorylation may not be implicated in activation of PLC-6 isozymes. Actually, our study has shown that genistein, a potent tyrosine kinase inhibitor, did not affect InsP levels obtained by thrombin stimulation in PLCS30 cells (data not shown).
Several lines of evidence are accumulating to support the view that activation of PKC attenuates receptor-mediated PLC activation in many types of cells by its negative feedback action (48,49). The target of PKC can be receptor, G-protein, or PLC itself. Phosphorylations by PKC of epidermal growth factor receptor and PLC-yl were proposed to explain the attenuation mechanism of phosphoinositide hydrolysis (50, 51). PLC-p1 also appears to be a target for the PKC-mediated attenuation in certain cells (2,38). Pretreatment with TPA of cells containing PLC-pl, PLC-yl, and PLC-61 elicited large increases in phosphorylation of serine residues in PLC-p1, but only small increases in PLC-yl and no effect in PLC-61, but does not affect its catalytic activity (38). It was also suggested that phosphorylation of PLC-p1 by PKC may interfere with its binding to Gqa (38). We demonstrated in PLC630 cells that PKC activation by TPA inhibited the thrombin-stimulated InsP production, but the level of the inhibition was almost similar to the vector-transfected (Vl) cells. Furthermore, the TPA pretreatment was without effect on InsP formation induced by GTPyS stimulation in both cells. In HEL cells, PKC activation was reported to cause loss of responsiveness to thrombin, and its inhibition was thought to be attributed to blockage of the Ca2+ entry by phosphorylation of thrombin receptor (52). However, it is not the case with PLCF30 cells, because the suppression of thrombin-stimulated InsP generation by TPA was not abolished by increasing the Ca2+ concentration. Therefore, we assume that phosphorylation of thrombin receptor by PKC activation in the V1 and PLC630 cells suppresses PLC activation by interfering with binding of the receptor with G-protein(s).
Leonis and Silbert (26) have recently shown that two CCL fibroblast mutant cell lines, which lack PLC-61 isozyme, showed significant differences in thrombin-stimulated PI turnover, decreased and increased responses, and also that transfection of the defected mutant with rat brain PLC-61 cDNA did not correct the agonist-induced PI turnover. We have observed in the present study that the increase of thrombin-stimulated InsP formation in the PLC630 cells was entirely dependent on extracellular Ca2+. Removal of extracellular Ca2+ in PLC630 diminished the marked response of thrombin-stimulated InsP formation. Therefore, it is conceivable that the CCL39 mutants have an additional mutation(s1, except for lack of PLCF1, for example, defective Ca2+ influx-related component. However, for elucidation of the precise mechanism of PLC-61 activation, further work, including a reconstitution experiment, should be required and is currently under progress in our laboratory.