BmDJ-1 Is a Key Regulator of Oxidative Modification in the Development of the Silkworm, Bombyx mori

We cloned cDNA for the Bombyx mori DJ-1 protein (BmDJ-1) from the brains of larvae. BmDJ-1 is composed of 190 amino acids and encoded by 672 nucleotides. Northern blot analysis showed that BmDJ-1 is transcribed as a 756-bp mRNA and has one isoform. Reverse transcriptase (RT)-PCR experiments revealed that the BmDJ-1 was present in the brain, fatbody, Malpighian tubule, ovary and testis but present in only low amounts in the silkgland and hemocyte of day 4 fifth instar larvae. Immunological analysis demonstrated the presence of BmDJ-1 in the brain, midgut, fatbody, Malpighian tubule, testis and ovary from the larvae to the adult. We found that BmDJ-1 has a unique expression pattern through the fifth instar larval to adult developmental stage. We assessed the anti-oxidative function of BmDJ-1 using rotenone (ROT) in day 3 fifth instar larvae. Administration of ROT to day 3 fifth instar larvae, together with exogenous (BmNPV-BmDJ-1 infection for 4 days in advance) BmDJ-1, produced significantly lower 24-h mortality in BmDJ-1 groups than in the control. 2D-PAGE revealed an isoelectric point (pI) shift to an acidic form for BmDJ-1 in BmN4 cells upon ROT stimulus. Among the factors examined for their effects on expression level of BmDJ-1 in the hemolymph, nitric oxide (NO) concentration was identified based on dramatic developmental stage-dependent changes. Administration of isosorbide dinitrate (ISDN), which is an NO donor, to BmN4 cells produced increased expression of BmDJ-1 compared to the control. These results suggest that BmDJ-1 might control oxidative stress in the cell due to NO and serves as a development modulation factor in B. mori.


Introduction
The protein DJ-1 is ubiquitously expressed in cells and it is highly conserved across a wide variety of organisms, showing moderate sequence identity with heat shock protein 31 (HSP31) chaperones and ThiJ/PfpI cysteine proteases [1]. Mutated forms of DJ-1 are known to cause early onset autosomal recessive juvenile Parkinson's disease (PD), and many studies have demonstrated a neuro-protective role of DJ-1. DJ-1, which is encoded by PARK7, is a multi-functional protein that plays roles in chaperoning, RNA-binding, SUMOylation, apoptosis, and protease activity [2].
Additionally, DJ-1 is induced by oxidative modification and is rapidly oxidized at position Cys 106 [3]. Oxidative modification leads to mitochondrial damage in cultured cells exposed to 1methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP), 6-hydroxydopamine (6-OHDA), paraquat (PQ), and rotenone (ROT), which inhibit the mitochondrial electron transfer chain of mitochondrial complex I [4]. These compounds enhance production of reactive oxygen species (ROS) and reduce production of ATP, resulting in mitochondria dysfunction [5]. DJ-1 seems to directly scavenge free radicals from mitochondria in response to these oxidative stresses. MPTP, 6-OHDA, PQ, and ROT are used to produce PD models in rats and Drosophila and to analyze the pathology of PD [6,7].
DJ-1 has a dimer structure, and the L166P mutation produces structural perturbation that causes the protein to be ubiquitinated and susceptible to degradation by the 26S proteasome, significantly reducing its half-life in vivo [8,9]. L166P DJ-1 forms unstable dimers with disrupted protein folding and function [10]. The C106A mutation results in a loss-of-function of DJ-1 protease and chaperone activity [11,12]. However, the precise pathology due to mutations and species-specific biological functions of DJ-1 remain unclear.
The silkworm, Bombyx mori, a Lepidopteran insect, has been utilized as a model system for basic science research because of its well-characterized genome, availability of various genetic mutants, and the development of transgenic, RNAi, and microarray technologies [13,14,15,16,17]. The complete silkworm genome has approximately 18,510 genes, including a substantial number of mammalian orthologs [13,16].
In the present study, we cloned the silkworm B. mori DJ-1 ortholog (BmDJ-1), clarified its expression pattern during development, and examined its anti-oxidative function. BmDJ-1 is a newly identified member of the DJ-1 family and is a growthassociated protein that is altered with development in B. mori.

Molecular cloning of BmDJ-1
Amplifying BmDJ-1 by RT-PCR with 59 RACE using genespecific primers from B. mori larvae brain cDNA produced an 86bp product. The Kozak consensus sequence AAAATGAAG [18] was found to be present at the site of translation initiation determined using NetStart software [19]. Therefore, we determined that the cDNA encoded a putative 59-untranslated sequence of 95 bp, an ATG start site, and an open reading frame (ORF) at position 96 extending to position 668. The deduced ORF of BmDJ-1 was composed of 672 nucleotides comprising 190 amino acids, had a molecular weight of 20,113 Dalton, and a putative isoelectric point (pI) of 5.15.
The nucleotide sequence reported in this paper has been submitted to GeneBank/DDBJ SAKURA Data bank Accession No. AB281053.
A computer search of the SMART database (http://smart. embl-heidelberg.de/) revealed that BmDJ-1 contained a DJ-1_PfpI domain at position 31T-173T. We identified the location of the BmDJ-1 gene in scaffold 2995719-2998746 of chromosome 23 at the splitting of 5 blocks by linkage mapping 28 chromosomes by SNP markers [20].

BmDJ-1 mRNA is expressed in various tissues in fifth instar larvae
Northern blot analysis revealed that there is a single transcription product for BmDJ-1 with a size of 756 bp ( Fig. 2A).

Specificity of antibody against BmDJ-1
We examined the utility of anti-BmDJ-1 antibodies raised against the recombinant Xpress-tagged BmDJ-1 to identify BmDJ-1. Anti-BmDJ-1 antibody reacted with both recombinant BmDJ-1 protein as a 25-KDa band and BmDJ-1 in the cell and tissue lysate from B. mori as a 20-KDa band. In contrast, BmDJ-1 antibodies did not recognize recombinant carotenoid binding protein (CBP) from B. mori tagged with GST [21] and HEK 293 cell lysate (Fig. 3,  lanes 1 and 4). The molecular weight of the recombinant BmDJ-1 protein (Fig. 3, lanes 2 and 3) was slightly greater than the endogenous BmDJ-1 protein (Fig. 3, lanes 5 and 6), excluding the possibility of non-specific binding to the Xpress tag.
Identification of developmental stage and tissue-specific expression patterns of BmDJ-1 by immunoblotting Distribution of BmDJ-1 expression by developmental stage and tissue is shown in Figure 4. Whole body expression is roughly equal for all larval instars, pupae, and adults ( Fig. 4A and S1A, lanes 1-7). Moreover, equal amounts of BmDJ-1 are found in the brains of fifth instar larvae, pupae, and adults, but it is slightly increased in larvae ( Fig. 4B and S1B, lanes 8-10). To determine the distribution pattern of BmDJ-1, we studied tissues (midgut, fatbody, Malpighian tubule, ovary, and testis; Fig. 4C and S1C) from day 0 fifth instar larvae to adults by immunoblotting. BmDJ-1 was expressed in the larval through adult developmental stages in these tissues, but expression was low in day 1 pupae (fatbody, Malpighian tubule and ovary; Fig. 4C, panels b, c, e, lane 14). Expression levels increased with pupal stadium from day 0 to 4 ( Fig. 4C, panels f-h) and high levels of BmDJ-1 expression were also identified in the testis during these developmental stages (Fig. 4C, panel q). Therefore, BmDJ-1 showed a unique day-today expression pattern from day 0 fifth instar larvae to the adult developmental stages.

The pI of BmDJ-1 shifted acidic by ROT stimulation
Treatment of BmN4 cells with 50 mM ROT produced a shift in the pI to acidic, as shown on 2D-PAGE and immunoblotting (Fig. 5).

BmDJ-1 overexpression in larvae causes resistance to ROT
ROT was used to produce an oxidative stress in order to examine the effect of exogenous BmDJ-1 protein. We determined the lethal dose (LD) of ROT for day 3 fifth instar larvae of 10.1 mg/g (LD 50 ; 95% CI, 6.02-17.4) (Fig. 6). Based on computer simulations of reactivity using SAS software, we determined the optimal ROT concentration for further testing of the protective effects of BmDJ-1 to be 20 mg/g.
We also confirmed virus-derived BmDJ-1 expression levels in the fatbodies of several insects after 1 day (24 h) and 4 days (day 4 fifth instar larvae) by RT-PCR and after 4 days by immunoblotting. Expression of blank-vector recombinant virus was detected as a 300bp band, while virus-derived BmDJ-1 expression was detected as an 850-bp band. The BmDJ-1 expression was absent at 24 h but was detected at 96 h (Fig. 7A). Virus-derived BmDJ-1 protein was expressed at about 2-fold greater levels in non-infected groups after 4 days, and BmDJ-1 protein expression in blank virus-infected control groups was significantly decreased ( Fig. 7B and S2).

Expression of BmDJ-1 and NO concentration
The expression pattern of BmDJ-1 was tissue-specific, reflecting the unique responses to oxidative stress. We examined some factors that might affect expression. NO concentration in the hemolymph was found to fluctuate from the fifth instar larva to adult (Fig. 8A), with high levels for day 0 and 6 fifth instar larvae and adults and gradually increasing NO concentration in the pupal stages. To test whether NO affects the expression of BmDJ-1, BmN4 cells were treated with 100 mM ISDN as an NO donor for 16 h. BmDJ-1 was detected in each sample by SDS-PAGE and immunoblotting with NO concentration (Fig. 8B) and BmDJ-1 expression ( Fig. 8C and S3) increased compared to the control (0.1% ethanol).

Discussion
Throughout its evolutionary history, DJ-1 shows a highly conserved amino acid sequence. Characterization of the B. mori variant, BmDJ-1, by cDNA cloning from the brains of the fifth instar larvae shows the presence of Cys and Leu, which are key residues for the function of DJ-1.
On a phylogenetic tree of DJ-1 proteins, two orthologs of D. melanogaster, DJ-1a and DJ-1b, and BmDJ-1 placed in distinct clusters. D. melanogaster DJ-1a is most highly expressed in the testis from the pupal stages to adult, and DJ-1b is expressed in almost all tissues from embryo to adult. Loss-of-function DJ-1b mutant flies are sensitive to oxidative modification from H 2 O 2 and paraquat, although the role played by DJ-1a remains unclear [22]. Thus, these two D. melanogaster DJ-1s appear to have distinct functions.
In contrast, BmDJ-1 exists as a single isoform based on the single 756-bp transcript and only one band for BmDJ-1 on northern blot assay. The EST database (SilkBase; http://morus. ab.a.u-tokyo.ac.jp/cgi-bin/index.cgi) shows two distinct EST clones (data not shown). While BmDJ-1 may exist as several kinds of splice variants, this could not be clarified in this study.

BmDJ-1 demonstrated resistance to oxidative stress by ROT
DJ-1 has been reported to play a role in anti-oxidative stress by several independent groups. We confirmed that BmDJ-1 changes to an acidic form that is affected by ROT treatment in BmN4 cells (Fig. 5), indicating a response to oxidative stress.
In exogenous tests of BmDJ-1 with ROT, the mortality rate of individuals with BmDJ-1 is significantly decreased in the presence of ROT treatment, while the control groups remain extremely sensitive. It has been reported that the start of protein synthesis for BmNPV is 24 h after infection and that the protein expression level peaks at 96 h [23]. Endogenous protein synthesis stopped at 24 h. Immunoblotting at 96 h showed that virus-derived BmDJ-1 protein expression was significantly increased and virus-infection control group of BmDJ-1 protein expression was significantly decreased. Our findings of virus-derived BmDJ-1 expression after 96 h corroborate those results and suggest that BmDJ-1 overexpression improves the survival of silkworm larvae treated with ROT.      BmDJ-1 expression controlled with NO BmDJ-1 showed a tissue-specific expression pattern that indicates unique responses to oxidative stress. We found that NO was an oxidative stressor in B. mori that could be modulated by BmDJ-1.
The BmDJ-1 expression pattern in tissues in this study suggested that BmDJ-1 expression correlates to the hemolymph NO concentration, which showed day-to-day fluctuation from fifth instar larvae to adult (Figs. 4C and 8A). Moreover, the expression of BmDJ-1 was increased and the pI shifted acidic due to exposure to an NO donor (data not shown). These results showed that BmDJ-1 was oxidized and its expression was regulated by NO.
Choi et al. [24] reported that the nitric oxide synthase (NOS) gene in B. mori shows the highest expression in Malpighian tubule in day 7 fifth instar larvae, suggesting that NO might be related to B. mori metamorphosis. Inoue et al. [25] reported that administration of ISDN, an NO donor, to the beetle Homoderus mellyi parry rapidly progresses pupation. Conversely, the administration of carnitine, which suppresses apoptosis of cells in larval beetles, extended the larval developmental period and generated huge adult beetles. These observations implicate NO in the mechanism of metamorphosis as an apoptosis initiator, though the underlying process remains unclear.
In B. mori, apoptosis is the principal mechanism for dynamic remodeling of the body structure during metamorphosis. Apoptosis mainly occurs during the pupal developmental period, during which the restructuring produces the adult body. In our observation, the increased expression levels of BmDJ-1 occur in the pupal developmental stage, which coincides with apoptosis and the apparent melting of the body. BmDJ-1 might be involved in the elimination of NO in metamorphosis.
Although DJ-1 protein acts as a controller caspase activation to alter self expression level in the apoptotic pathway [26], we cannot determine a direct relationship between BmDJ-1 and NO generation in metamorphosis based on these experiments.
In future studies, we will investigate whether BmDJ-1 directly regulates NO in metamorphosis.

Ethics statement
The study protocol for the experimental use of the animals was approved by the Ethics Committee of Meiji Pharmaceutical University (Approval ID 2004).

Insects
The hybrid strain Kinshu x Showa was supplied from Ueda-Sha Co. Ltd., Nagano, Japan. Individuals were reared on the artificial diet Silkmate 2S (NOSAN, Tsukuba, Japan) and kept at 25uC on a 12 h light/12 h dark daily cycle.

Molecular cloning of BmDJ-1
We first searched the B. mori expressed sequence tag (EST) database on KAIKOBLAST (kaikoblast.dna.affrc.go.jp) using the Drosophila melanogaster DJ-1 alpha (NM_137072) or beta (NM_143568) sequence as a query, and identified the EST clone NRPG1136, which did not overlap the 59 end of the coding region of the B. mori DJ-1 gene (BmDJ-1). The entire coding sequence was determined using total RNA extracted from the brains of day 3 fifth instar larvae by an RNeasy mini kit (Qiagen, Valencia, CA, USA). DNase-treated total RNA was processed for cDNA synthesis using oligo(dT)12-18 primers and SuperScript II reverse transcriptase (Invitrogen), and cDNA was amplified by PCR using Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA, USA) and the primers 59-TCAAGAACAATGAGCAAGTCTGCG-39 and 59-TAATATTAGTACTGCGAGATTAAC-39. The amplified products were cloned into a cloning vector p3T (MoBiTec, Göttingen, Germany). The purified vectors were processed for sequencing by the dideoxynucleotide chain termination method on an ABI PRIZM 3100 Genetic Analyzer (Applied Biosystems, Tokyo, Japan). The cDNA clone, NRPG1136, was provided by the National Bioresource Project (MEXT, Japan).

59-Rapid Amplification of cDNA ends
The 59-terminal cDNA ends were amplified using the SMART RACE cDNA Amplification kit (Clontech, Mountain View, CA, USA) according to the supplier's instructions with primers 59- GCCAGCTAGAGTAACTGTTACCCC-39 and 59-AGTCAC-TTGCCTTGAGCACAGCAC-39. The amplified products were cloned into a p3T vector for sequencing.

Recombinant protein
The ORFs of BmDJ-1 were amplified by PCR using PfuTurbo DNA polymerase and primers 59-AGCAAGTCTGCGTTAGT-GAT-39 and 59-TTAGTACTGCGAGATTAACA-39. Products were cloned into a prokaryotic expression vector pTrcHis-TOPO with a TOPO TA cloning kit (Invitrogen) and expressed in E. coli as fusion proteins with N-terminal Xpress tags. The nucleotide sequence was confirmed by sequencing. Recombinant BmDJ-1 expressed in E. coli was purified with HIS-Select spin columns (Sigma, St. Louis, MO, USA) according to methods described previously [27]. A recombinant b-galactosidase (LacZ) fragment tagged with X-press included in the TOPO TA cloning kit was used as a negative control.

Immunology
The antibody for immunoblotting was raised in Japanese white rabbits by subcutaneous injection of the recombinant BmDJ-1 and Ribi adjuvant system (Corixa Co., Hamilton, MT, USA) mixture. The serum was stored at 280uC.

Immunoblotting
To identify the presence of BmDJ-1 in different tissues and cells, protein samples (5 mg) were separated on SDS-PAGE, transferred to nitrocellulose membranes using the method of Towbin et al. [28], and immunoblotted using rabbit anti-BmDJ-1 antibody and goat anti-rabbit IgG-conjugated horseradish peroxidase (HRP). The membranes were developed using a chemiluminescent substrate (Pierce, Rockford, IL, USA).
The tissue distribution of BmDJ-1 was determined for the midgut, fatbody, Malpighian tubule, testis, and ovary from day 0 fifth instar larvae, pupae, and adults. Each tissue sample was run on the same gel, which was also loaded with 20 ng of recombinant Xpress-tagged BmDJ-1. The distribution of BmDJ-1 from first to fifth instar larvae, pupae and adult on the whole body and brain of larvae, pupae and adults were also determined. All tissues were homogenized in RIPA lysis buffer composed of 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% SDS, and a cocktail of protease inhibitors (Sigma), followed by centrifugation at 10,0006 g for 15 min.
The protein concentration was determined by a Bradford assay kit (Pierce). Samples of supernatant (5 mg of protein) were separated by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with anti-BmDJ-1 antibody following the procedure described above.

Northern blot analysis
Total RNA derived from the ovaries of day 4 fifth instar larvae were used. Total RNA (12 mg) was separated on a 1.5% agarose-6% formaldehyde gel and transferred to a nylon membrane. DIGlabeled probes were synthesized using the PCR DIG probe synthesis kit (Roche Diagnostics, Mannheim, Germany) according to the supplier's instructions with the primers 59-CATTT-GTGCTGCTTCCATAGCGTT-39 and 59-CATTCCCTTTT-CGACTTGATCGGC-39. After pre-hybridization, the membranes were hybridized with the DIG-labeled probes at 54uC overnight. The specific reaction was visualized on Kodak X-OMAT AR X-ray films by the DIG chemiluminescence detection kit (Roche Diagnostics).

RT-PCR
Total RNA derived from the brain, midgut, fatbody, Malpighian tubule, testis, ovary, and hemocyte of day 4 fifth instar larvae was DNase-treated and processed for cDNA synthesis using oligo(dT)12-18 primers and SuperScript II reverse transcriptase (Invitrogen). cDNA was amplified by PCR using Taq DNA polymerase (Qiagen) and the primers 59-CATTTGTGCTG-CTTCCATAGCGTT-39 and 59-CATTCCCTTTTCGACTT-GATCGGC-39. Amplification was carried out for 30 cycles of denaturing for 40 s at 94uC, annealing for 40 s at 50uC and extension for 90 s at 72uC. Amplified PCR products were separated by agarose gel, stained with ethidium bromide, and visualized under UV light.

Transfer plasmid and generation of recombinant virus
The ORF sequence of BmDJ-1 was amplified by PCR from brain cDNA as described above, with the primers 59-GGG-GTACCCCATGAGCAAGTCTGCGTTAGTGAT-39 and 59-GGAATTCCAATATTAGTACTGCGAGATTAAC-39.
The amplified region was digested with EcoRI and KpnI and cloned into the baculovirus transfer pBK283 vector. Blank pBK283 vector was used as a control. For generating recombinant BmNPV, we used a Bom-EX kit (NOSAN) according to the supplier's instructions. The recombinant BmNPV nucleotide sequence was confirmed by sequencing using the primers 59-ACTGTCGACAAGCTCTGTCC-39 and 59-ACAACGCACA-GAATCTAACGC-39. Purified recombinant virus was titrated by plaque assay, and high titer stocks (2610 7 pfu/ml) were used for infecting larvae.

Determination of LD 50 of day 4 fifth instar larvae by ROT stimulation
To determine the LD 50 of day 3 fifth instar larvae by ROT (Sigma) stimulation, we injected ROT intrahemocoelically to larvae weighing 3.5 to 4.0 g using a disposable syringe (Terumo, Tokyo, Japan) with a 30G needle. ROT was dissolved in DMSO (prepared immediately before use and stored in the dark) at 0, 1.25, 2.5, 5.0, 10, 20, 40, and 80 mg/g and injected into larvae in a volume of 10 ml/g body weight. The number of dead silkworms after 24 h was counted and the mortality rate (%) = (X/Y)6100 was calculated, where X = dead larvae in the group and Y = total larvae in the group. The mortality rates were analyzed with Probit analyses [29] using the Probit Analysis option in the SAS 8.2 software package (SAS Institute Japan Ltd., Tokyo, Japan) to calculate the LD 50 .
Overexpression of BmDJ-1 to larvae and exposure to ROT oxidative stimuli A 50 ml aliquot of BmNPV-BmDJ-1 or BmNPV-blank-vector (1610 5 pfu/larva) was injected intrahemocoelically into day 0 fifth instar larvae using a disposable syringe (Terumo) with a 30G needle. Blank-vector recombinant virus was injected as a control.
After rearing for 4 days on an artificial diet, larvae were examined for overexpression of BmDJ-1 to assess protection from oxidative stress due to ROT.
Virus-derived BmDJ-1 expression level was measured in the dissected fatbodies of several insects after 4 days (day 3 fifth instar) by immunoblotting.
We surmised the ROT dose that would be most effective in the experimental model with exogenous BmDJ-1 based on a report of the administration of exogenous DJ-1 [30].
ROT, prepared at 20 mg/g (LD 70 ), was injected to three groups of 10 to 20 larvae in a volume of 10 ml/g body weight. The number of dead silkworms after 24 h was counted and the mortality rate (%) was calculated.
Data were analyzed with the multiple comparison test followed by the Cochran-Armitage test for dose-response relationship and Steel's (non-parametric) multiple comparison test. P,0.05 was considered significant. All statistical analyses were carried out using SAS system 8.2 software. Three trials were performed in each experiment.
BmN4 cells treated with ROT, two-dimensional (2D) gel electrophoresis, and detection of BmDJ-1 BmN4 cells (2610 6 ) were grown on 6-well Falcon plates (BD Biosciences, Franklin Lakes, NJ, USA) and washed twice with PBS followed by 3 h of treatment with TC-100 medium containing ROT (50 mM) dissolved in 0.1% DMSO or 0.1% DMSO as a control in the dark. To prepare total protein extracts for twodimensional (2D) gel electrophoretic analysis, the cells were sonicated in rehydration buffer comprising 8 M urea, 2% CHAPS, 0.5% carrier ampholytes at pH 3-10, 20 mM dithiothreitol, 0.002% bromophenol blue, and a cocktail of protease inhibitors. Urea-soluble proteins were separated by isoelectric focusing (IEF) using the ZOOM IPGRunner system loaded with an immobilized pH 3-10 gradient strip (Invitrogen), as described previously [16]. After the first dimension of IEF, the protein was separated in the second dimension on a 4-12% NuPAGE polyacrylamide gel (Invitrogen). For detection of BmDJ-1, the gel was transferred to a polyvinylidene difluoride (PVDF) membrane for immunoblotting.
All incubation steps were carried out at 25uC in the dark. Three trials were performed for each experiment.

Collection of samples and measurement of NO levels
Hemolymph (250 ml) was collected from day 0 fifth instar larvae, pupae and adults, or from medium to measure the concentration of NO. To remove proteins, samples were mixed with methanol (2:1 by volume), followed by centrifugation at 10,0006 g for 20 min, and NO levels in the supernatants were measured using an NOx analyzer (ENO-20; Eicom, Kyoto, Japan), according to the manual.
BmN4 cell treatment with ISDN and detection of BmDJ-1 BmN4 cells (2610 6 ) were grown on 6-well Falcon plates (BD Biosciences) and washed twice with PBS followed by 16 h of treatment with TC-100 medium containing 100 mM of isosorbide dinitrate (ISDN; prepared immediately prior to use and kept in the dark) dissolved in 0.1% ethanol or with 0.1% ethanol alone as a control. Total protein extracts were prepared for immunoblotting and culture medium was prepared for NO analysis. Statistical analysis was performed using Student's t-test. P,0.05 was considered significant. All statistical analyses were carried out using SAS system 8.2 software. Three trials were performed for each experiment.