The Lean fish showed an enhanced response to low dietary n

The Lean fish showed an enhanced response to low dietary n ceritinib novartis 3 LC PUFA and the expression of 5fad, 6fad, elovl5b and elovl2 in the intestine showed high plasticity Inhibitors,Modulators,Libraries and was reflected in tissue biochemical com position indicating that their transcriptional regulation might be under feedback control by n 3 LC PUFA, mainly DHA. Lower n 3 LC PUFA in VO increased lipo genesis in Lean salmon, Inhibitors,Modulators,Libraries assessed by expression of FAS, while B oxidation appeared unaffected, although tran scripts involved in mitochondrial respiratory or electron transport chains were down regulated, suggesting reduced activity in fish fed VO. Higher expression of genes and proteins involved in xenobiotic metabolism, antioxidant defence, and apoptosis were observed in VO fed fish, suggesting they might be responding to higher levels of contaminants, particularly PAH, in the diet.

However, the intestine appeared able to metabolize and detoxify Inhibitors,Modulators,Libraries xenobiotic substances potentially present in the Inhibitors,Modulators,Libraries diet without major deleterious effects. Nonetheless, the data suggest that further attention should be given to contaminants in VO in the future. On the other hand, the data indicate potential genotype specific differences in the response of the intestinal transcriptome and proteome to dietary VO. These include potential changes in structural properties of the intestinal layer and defence against cellular stress suggesting the Lean group was more susceptible to diet induced oxidative stress.

Considering the possibility of selecting families better adapted to alternative diet formulations, and the central role of intestine as a major barrier to nutrients, contaminants and pathogens, greater attention should be given to this organ when evaluating the effects of diet and genotype. Methods Feeding Inhibitors,Modulators,Libraries trial and sampling A dietary trial was conducted using two genetically char acterized groups of Atlantic salmon post smolts comprising full sib families selected from a breeding program. The choice of the family groups was based on estimated breeding values of the par ents for high or low flesh adiposity, assessed by Torry Fatmeter, a trait that was found to have a heritability ranging from 0. 17 to 0. 39 in this dataset. The two groups were created from four unrelated full sib families, two families from the extreme lower end of the EBV distribution for flesh lipid content and two families from the extreme upper end of the distribution.

The average EBV for the lipid content of the Fat families was 2. 00 percentage units higher than that of the Lean families, representing a standardised selection differential of 2. 33 standard deviations. Assessment of the flesh and visceral lipid contents at the end of the trial confirmed differences in adiposity between the groups. Two thousand fish of each group were stocked into eight 12 x 5m3 net pens at the Ardnish Fish Trials Unit.

The libraries were loaded onto flow cell channels for sequencing

The libraries were loaded onto flow cell channels for sequencing on the Illumina HiSeq 2000 in strument by the Chinese National Human Genome Cen ter. A total Dovitinib structure of six paired end cDNA libraries of zebrafish livers were constructed for each of the test groups of WED immunized and mock immunized fish. Triplicate biological replicates were per formed for each group. Raw data were deposited in the NCBI database under submission num ber SRA048658. 2. Transcriptome analysis The Illumina HiSeq 2000 system generated 120 bp raw PE reads were first processed by the FASTX Toolkit to remove the reads with sequencing adaptors and of low quality. Then, the Burrows Wheeler Aligners Smith Waterman Alignment Inhibitors,Modulators,Libraries program was used to align the remaining reads to the reference zebrafish mRNA from the Ensembl database.

The transcription level of each gene was deduced by deter mining the total number of reads mapped to each gene using Picard tools. Dif ferentially expressed genes were identified by the DESeq package in R software, using two fold change 1 or 1 and p value 0. 05 as the threshold. After data normalization by the p Inhibitors,Modulators,Libraries value and FDR calculation, the resulting expression intensity values were analyzed by the MA plot based method, as described by Wang et al. Functional analysis of differentially expressed genes The Database for Annotation, Visualization and Inte grated Discovery was used to investi gate functional enrichment Inhibitors,Modulators,Libraries for over and under expressed genes by more than two fold in the WED immunized group relative to the mock immunized group.

Gene functional enrichment was performed using the default parameters Inhibitors,Modulators,Libraries in DAVID to obtain an adjusted p value 0. 05 for the test gene group versus the zebra fish gene ontology annotation set. The fold enrichment cut off suggested for DAVID functional annotation is 1. 5. In addition, the significantly up regulated genes from the differentially expressed genes dataset were further analyzed by Inhibitors,Modulators,Libraries investigating the corresponding GO biological processes. Furthermore, GO analysis of genes transcribed at different levels was also performed using the Biological Networks Gene Ontology tool, which is based on the Cytoscape software. The hypergeometric test with Benjamini Hochberg False Discovery Rate was performed using the default parameters to obtain an adjusted p value between the test gene group and the merged non redundancy zebrafish and mouse GO annotation set. Finally, the web based Kyoto Encyclopedia of Genes and Gen omes pathway analysis program run by the KEGG certainly Automatic Annotation Server was used to obtain func tional annotation of genes by performing basic local alignment search tool mediated comparisons against the manually curated KEGG GENES database.

Cellular debris was removed by centrifugation at 12,000 g for 30

Cellular debris was removed by centrifugation at 12,000 g for 30 minutes at 4 C. The supernatants were incubated with anti GFP antibodies overnight at 4 C. After incubation, protein G Sepharose was used for precipitation. The beads were washed with TSPI buffer four times and then eluted with SDS sample buf fer for immunoblot analysis. selleck chemicals Pazopanib Statistical analysis Densitometric analysis of immunoblots from three inde pendent experiments was performed using ImageJ windows version. The data were analyzed using windows version of Origin 6. 0 or Prism 5. The pictures in Figure 1A were draw using DOG 1. 0. Background TRA1 is an essential gene in Saccharomyces cerevisiae that encodes a 437 kDa protein product.

Inhibitors,Modulators,Libraries It is a member of a family of key signaling and regulatory Inhibitors,Modulators,Libraries molecules that con tain a C terminal phosphatidylinositol 3 kinase domain and is a Inhibitors,Modulators,Libraries component of two multisubunit tran scriptional regulatory complexes, the SAGA SLIK and NuA4 complexes, which also contain the histone acetyl transferase enzymes, Gcn5 and Esa1, respectively. Tra1 interacts directly with transcriptional activator pro teins and is thought to be critical in recruitment of SAGA SLIK and NuA4 to their target promoters. Previously we identified mutations in the C terminal PI3K domain of Tra1 that showed defects in transcriptional activation, sensitivity to ethanol and the cell wall destabi lizing agent calcofluor white and resulted in shortened tel omeres. The pattern of changes neither fully mimicked those seen upon disruption of other SAGA SLIK nor NuA4 components.

For example, unlike strains with deletions of NuA4 components, the tra1 mutant Inhibitors,Modulators,Libraries strains were relatively insensitive to DNA damaging agents. We performed an initial systematic genetic array analysis with the most pronounced allele, tra1SRR3413. This analysis did not identify any synthetic lethal Inhibitors,Modulators,Libraries interactions but did reveal 23 synthetic slow growth interactions, many in combination with deletions of genes involved in cell membrane wall processes. As the lack of synthetic lethal interactions may have arisen from an incomplete selection against diploids in the automated screens, we repeated the screen in a strain background that selects more strongly against dip loids. This analysis identified 114 genes displaying syn thetic sick lethal interactions with tra1SRR3413.

Genes involved in transcription, RNA processing, selleck chem inhibitor mitochondrial function and membrane sorting protein trafficking were prevalent. The phenotypes and genetic interactions of these strains also point to a role for Tra1 in the cellular response to stress. Results SGA analysis of tra1SRR3413 As a component of both SAGA and NuA4 complexes, Tra1 is positioned to play a major role in nuclear processes. Previously we performed an SGA analysis on Tra1 using an allele that partially impairs function. No synthetic lethal interactions were obtained in this analysis.

However a consistent genetic and genomic analysis of processes af

However a consistent genetic and genomic analysis of processes affecting pollen viability is currently non existent. selleck bio The pollen development in Prunus species and other woody perennial plants from temperate climates such as apple and poplar is affected by the seasonal cessation of meristem growth termed endodormancy. Endodormancy contributes to elude the detrimental effects of the low temperatures in winter by preventing the resumption of growth under non optimal conditions for survival. The growth inhibition of endodormant buds is due to internal signals within the buds, in contrast to growth inhibition by other distal organs, or by environmen tal factors. For the purpose of this work we have employed the term dormancy to refer to the endodormant state.

In these species, the flower Inhibitors,Modulators,Libraries buds start to differentiate in summer and continue their reproductive development until growth is arrested in autumn. After a period of chilling accumulation required for dormancy release, pollen mother cells within the anthers initiate meiosis and further microspore development, resulting in fully mature pollen grains. In order to identify putative genes involved in tapetum function, pollen development and pollen wall formation in peach, we analyzed the results of two transcriptomic experiments comparing gene expression between dormant and dormancy released flower buds, and in peach cultivars with dif ferent dormancy behaviour. This work led us to postulate a role for several genes in sporopollenin synthesis and deposition, and transcriptional regulation of pollen development processes, based Inhibitors,Modulators,Libraries on expression analysis and previous works in model species.

Results and discussion Identification of genes up regulated in late stages of reproductive bud development Meristems Inhibitors,Modulators,Libraries of woody perennials from temperate climates go through the cold season in a dormant stage, pro tected into specialized structures named buds. In peach, reproductive buds are typically arranged in pairs, flanking a single vegetative bud. In suc cessive steps, flower buds are Inhibitors,Modulators,Libraries induced and differentiate in summer, and enter a dormancy period in autumn winter. The dormancy is released after a required chil ling period, whose length is genotype specific. Finally their reproductive organs resume growth and develop ment leading to blooming when temperature conditions become favourable.

In anthers, the release Inhibitors,Modulators,Libraries of dormancy initiates microsporogenesis, pollen develop ment and maturation. We previously studied the genome wide modification of selleck chemical Dovitinib gene expression in flower buds of peach through two complementary transcriptomic approaches. In the first work we isolated differentially enriched transcripts in dormant buds and dormancy released buds by the suppression subtractive hybridization procedure. SSH procedure relies on the selective amplification and enrichment of abundant cDNAs in a sample when incubated and hybridized with an excess of a refer ence sample.

To appreciate the reliability of the knowledge that the model pro

To appreciate the reliability of the knowledge that the model provides in terms of elucidating the impact of the modulation of P gp activity on drug distribution, we had access to WT and KO tissue concentrations of domper idone, an antiemetic drug associated with cardiac toxicity. The choice of this drug model was motivated by previous in vitro results, which suggested that domperidone could Pazopanib structure be highly transported by P gp. While this data set cannot be considered rich enough to validate the developed PBPK model, it can at least show that, the model simulations lie within realistic values by capturing points in the main strategic regions of the tissue concentration profiles, namely at the maximum concentration and the elimination phase.

Methods Structure of the PBPK model The present investigation Inhibitors,Modulators,Libraries focuses on P gp substrate dis tribution Inhibitors,Modulators,Libraries in heart and brain tissue where this transporter has a protective function. Our whole body PBPK model included these tissues as well as core tissues, organs and fluids, namely liver, arterial and venous blood, along with the adipose tissue because of its involvement in the disposition of lipophilic drugs. To make the model readily usable for subsequent updates and future experimental data, we also included bone, gut, lung, kidneys, muscle skin and spleen in the PBPK structure. The PBPK model is mathematically formulated as a set of ordinary differential equations of mass balance that represents the time dependent variation of the drug concentration in each tissue. We systematically Inhibitors,Modulators,Libraries performed an overall mass balance of the whole body PBPK model to assure that mass conservation laws are respected.

Tissue distribution models transporters such as influx transporters and additional efflux transporters. Well stirred model At this first step of Inhibitors,Modulators,Libraries model development, the whole body PBPK model Inhibitors,Modulators,Libraries is based on perfusion selleck Paclitaxel limited model of disposition. The uptake rate of the drug into tissues is limited by the flow rate to tissue rather than the diffusion rate across cell membranes. In this case, the unbound concentration of drug in tissue is in equilibrium with the unbound drug in the outcoming blood. The application of a WS model requires the tissue to plasma partition coefficient of each tissue included in the PBPK model as input parameters. By definition, these partition coefficients were calculated as The parameters used in the equations presented in this section refer to concentration, volume, blood flow to tissue, tissueplasma partition coefficient. bloodplasma ratio, unbound fraction of drug, clearance, and permeability surface area product.

This included transcripts

This included transcripts encoding for ion channel and trans porter proteins and regulators of synaptic transmission. These find ings suggest a profound and sustained impairment of neu ronal signal transmission during the course of EAE. The persistent suppression of transcripts for MOG, myelin basic protein, myelin associated oligodendrocytic basic protein and oligodendrocyte myelin glyco protein indicates dysfunction or even loss of oli godendrocytes, putative primary targets of the immune response in EAE. Upregulation of CNS genes in the recovery phase but not in the acute phase suggests a potential contribution to repair processes. Twelve CNS specific genes showed this expression profile. Some of these genes had been described to be involved in repair or regenerative processes within the CNS before.

Id4. Ednrb. Cdh22 and Glrx2. Interest ingly, however, the angiogenic growth factor PdgfdPnlip, the cytokinesis controlling armadillo protein Pkp4. the peroxysome regulator Pex11a and Hoxb8 have not been associated with repair processes in the CNS yet. Two transcripts of this group are Inhibitors,Modulators,Libraries not yet defined. The cholesterol biosynthesis pathway in EAE Cholesterol is an essential component of eukaryotic plasma membranes and represents around 30% of the lipid content within the CNS. In the mature CNS, the highest cholesterol content can be found in oligodendro cyte myelin. Cholesterol availability in oligodendrocytes is rate limiting for myelination. Interestingly, our data revealed a suppression of many genes Inhibitors,Modulators,Libraries associated with the cholesterol biosynthesis path way throughout the acute, recovery and relapse time points.

This suppression included HMG CoA reductase catalysing the rate limiting step in cholesterol biosynthe sis. A comparable general decrease of the expression of cho lesterol transport proteins was not noticed. Rather, Inhibitors,Modulators,Libraries apolipoprotein C I and CD36 were strongly upregulated during the acute disease phase. The expression profile and the known expression pattern of CD36 suggest that its upregulation is probably Inhibitors,Modulators,Libraries caused by As the transcription of the HMG CoA reductase or LDLR is inhibited by the presence of cholesterol derivatives, we assessed the total cholesterol concentration in the spinal cord tissue and detected no change Inhibitors,Modulators,Libraries of the cholesterol con centration at any time point of EAE.

Hence, the downregulation of the expression of the enzymes of the cholesterol biosynthesis pathway was not associated with a corresponding decrease of cholesterol concentra tion. SLPI in MOG induced EAE of DA rats The expression of the secretory leukocyte protease inhibi tor was approximately 100 fold upregulated dur ing the acute disease phase and more than 10 fold during the recovery and relapsing phases. Immuno histochemical analyses of spinal cord slices from rats suf fering from acute EAE showed a strong SLPI staining restricted to the inflammatory infiltrates.

After incubation, the noninvaded cells on the upper membrane surf

After incubation, the noninvaded cells on the upper membrane surface were removed with a cotton tip, and the cells that passed through the filter were fixed and stained using 0. 1% crystal violet. The sellckchem numbers of invaded cells were counted in five randomly selected high power fields under a microscope. This experiment was performed in triplicate. Matrigel in vitro HUVEC tube formation assay Cells Transfected with control or FoxM1 siRNA were cultured in serum free RPMI 1640 for 24 hours. The conditioned medium were collected, centrifuged and stored at 20 C until assay. HUVEC in 500 ul of the indicated conditioned medium were seeded onto a 24 well plate, which was precoated Inhibitors,Modulators,Libraries with 100 ul of growth factor reduced Matrigel for 30 minutes.

Following stimulation with the cell conditioned medium for 12 hours, Inhibitors,Modulators,Libraries tube formation was observed under an inverted microscope and counted. The number of tube formations was measured by counting the number of tube like structures formed by connected endothelial cells in five Inhibitors,Modulators,Libraries randomly selected fields under a microscope. The assay was performed in triplicate. Statistical analysis The statistical analyses were performed using the Statis tical Package for the Social Sciences, version 16. 0. A paired samples t test was used to compare FoxM1 mRNA and protein expression in the ccRCC tissues with that of their paired adjacent nontumor tissue samples. The relationship between FoxM1 protein expression and the clinicopathological features was analyzed using ��2 tests. Overall survival curves were calculated with the Kaplan Meier method and were analyzed with the log rank test.

A Cox propor tionalhazards analysis was used in univariate and multi variate analyses to explore the effects of FoxM1 expression and ccRCC clinicopathological variables on survival. Unpaired 2 tailed Students t tests were used to analyze comparisons between the 2 groups. A P value of 0. 05 was regarded as statistically significant. Results FoxM1 Inhibitors,Modulators,Libraries mRNA and protein expression in primary ccRCC tissue samples and RCC cell lines We first examined FoxM1 mRNA expression in 39 paired clinical samples from ccRCC patients by real time quantitative PCR. The results revealed a statistically significant elevation of FoxM1 mRNA in tumors, as compared to the matched adjacent nontumor tissues. To investigate whether FoxM1 was also elevated at the protein level, western blot was performed on those 39 paired ccRCC clinical samples.

We found that the protein level of FoxM1 in tumor tis sues was significantly higher than that in adjacent non tumor tissues, consistent Inhibitors,Modulators,Libraries with the results of real time quantitative PCR. The protein level of FoxM1 in four representative pairs of samples is shown in Figure 1C. We also used real time quantitative PCR and western blot to detect the expression of FoxM1 mRNA and protein in RCC cell lines as well as in an immortalized Sorafenib normal human proximal tubule epithelial cell line.

The vast majority of false positives were due to undetected heter

The vast majority of false positives were due to undetected heterozygous alleles in the germline. Somatic mutations were observed Inhibitors,Modulators,Libraries in two well characterized tumor suppressor genes, TP53 and a truncating mutation in RB1 removing 75% of its coding sequence, with TP53 also within a region of heterozygous loss. Inhibitors,Modulators,Libraries Transcriptome analysis Whole transcriptome shotgun sequencing was conducted to profile the expression of tumor transcripts. In the absence of an equivalent nor mal tissue for comparison, we compared expression changes to the patients leukocytes and a compendium of 50 tumor derived WTSS datasets, which would avoid spurious observations due to technical or methodologi cal differences between gene expression profiling plat forms.

This compendium approach allowed us to identify a specific and unique molecular transcript signa ture for this tumor, as compared to unrelated tumors, enriched in cancer causing events specific to the patients tumor and therefore should represent relevant drug targets for therapeutic intervention. Inhibitors,Modulators,Libraries There were 3,064 differentially expressed genes in the lung tumor versus the blood compendium. This analysis provided insight into those genes whose expression rate was likely to be a driving factor specific to this tumor, not identifying genes that correlate simply with proliferation and cell division. It is conceivable that such an approach, coupled with a greater understanding from multiple tumor datasets, could be replaced by the absolute quan tification of oncogene expression as a means to deter mine clinical relevance.

Changes in Inhibitors,Modulators,Libraries expression in both metastases were significantly associated with copy num ber Inhibitors,Modulators,Libraries changes. A large number of canonical pathways were identified as over represented in the pathway analysis. Specifically, ten pathways were significant from the lung versus blood compendium gene lists, two from skin versus blood com pendium, and 98 from skin versus lung. These included many molecular mechanisms of cancer and cancer related signaling pathways, such as mammalian target of rapamycin signaling, p53 signaling, Myc mediated apoptosis signaling, vascular endothelial growth factor signaling, phosphoinositide 3 kinase AKT signaling, and phosphatase and ten sin homolog signaling, amongst others. We correlated the mutated, amplified or differentially expressed genes with known cancer pathways from the Kyoto Encyclopedia of Genes and Genomes database and to drug targets present in the Drug Bank database.

The 15 amplified, over expressed or mutated genes in cancer pathways targetable by approved drugs are listed in Table S2 in Additional file 1. Some amplified genes, such as NKX3 1, RBBP8 and CABL1, were implicated in cancer but are not well char acterized in this role. In addition, they did not have Pacritinib FLT3 known drugs targeting them.

Interestingly, lower neutrophil count has been recorded in N can

Interestingly, lower neutrophil count has been recorded in N. caninum selleckchem seropositive cows. Further experiments should confirm the biological relevance of the BHB in this parasite infection. Lipid droplets, major lipid storage structures, are com posed of a triglyceride and cholesteryl ester core with a surrounding monolayer of phospholipid, cholesterol, and a variety of associated proteins with diverse functions in cell metabolism, signaling, and inflammation. The interaction of parasite proteins with these lipid bodies is important for the replication of IC, such as N. caninum and for the biogenesis of new parasite particles. Inter Inhibitors,Modulators,Libraries estingly, culture medium concentrations of TGAs and NEFAs significantly increased by N.

caninum infection, which is consistent with a previous Inhibitors,Modulators,Libraries report where the size and number of lipid droplets whose major component is TGAs increased in cells infected by a related protozoan, Toxoplasma Inhibitors,Modulators,Libraries gondii. The endogenously generated un saturated FAs may play a role in TGA accumulation, and it is possible that unsaturated FAs activate signaling pathways that promote TGA storage. Although infection increases cellular neu tral lipids, including TGAs, the cholesterol contents did not seem to be significantly affected by N. caninum infection. The marked increase in the concentrations of TGAs and NEFA in infected culture medium compared to con trol and the significant changes in expression of genes involved in lipid biogenesis demon strate that lipids play a very important role in the IC life cycle of N. caninum.

Increased Inhibitors,Modulators,Libraries lipid droplets formation and association with the parasitophorous vacuole has been demonstrated in infections by other parasites in cluding T. gondii, Trypanosoma cruzi, Leishmania amazonensis, Plasmodium falciparum, and P. berghei. However, to date the mecha nisms that govern lipid droplets biogenesis and its role to N. caninum pathogenesis are not known. Further, we determined the temporal changes in exo metabolome composition by obtaining Raman spectra from medium of infected and control cultures. PCA scores plots and loadings plots showed a clear separation between samples taken from infected and control cul tures, supporting the feasibility of this method for the investigation of the biochemical Inhibitors,Modulators,Libraries differences be tween control and infected cultures.

These data also in dicate that footrpinting metabolic analysis using label free Raman spectroscopic imaging combined with multivariate chemometric analysis has enough resolution to monitor infection related metabolic changes over the course of time spanning the IC life cycle of the parasite. This time resolved based analysis is essential since metabolic differ ences can be highly dependent Lapatinib order on growth phase of the cell, and cellular biochemistry changes during growth of both the cell and the parasite.

The reaction mixture and GSH control were analyzed in positive io

The reaction mixture and GSH control were analyzed in positive ion mode by electrospray ionization using a LTQ XL ion trap mass spectrometer. Real time screen shots of the chro matograms selleck chemicals llc were captured in the Xcalibur browser, ver sion 3. 3. Reduced GSH is known to produce a peak with a m z 308. QM Inhibitors,Modulators,Libraries covalently bound to gluta thione has a predicted peak at m z 413. 4 based on molecular weight calculations in Symyx Draw 3. 2. DNA ladder assay LNCaP and RWPE 1 cells were grown in 75 cm3 cell culture flasks to 80% confluence and treated with NPAA, staurosporine, or DMSO as previously described. Cells were collected Inhibitors,Modulators,Libraries as previously described and were then lysed and processed with the Apoptosis DNA Ladder Kit according to the manufacturers instructions.

The RWPE 1 and LNCaP sample DNA, the stauros porine control, and DNA ladder supplied with the kit were mixed Inhibitors,Modulators,Libraries with loading buffer and added to a 2% agarose gel containing 1 10,000 dilution of SYBR Safe. The gel was electrophoresed Inhibitors,Modulators,Libraries at 75 V for 2 hours in TBE buffer and then photographed under UV light using a ChemiDoc XRS system with Image Lab software. Protein carbonyl assay RWPE 1, LNCaP, COS 7, and COS 7 OPH cells were grown in 25 cm3 cell culture flasks to 80% confluence. The cells were then treated with NPAA or DMSO and collected as previously described. The cells were then lysed with 2% digitonin in PBS and the protein concentra tion was determined using the BCA assay kit. For each sample, an aliquot of 50 ul of protein lysate containing 5 ug ul of protein in PBS was added to two 1. 5 ml tubes. One tube was used as the negative control tube.

Inhibitors,Modulators,Libraries A volume of 200 ul of 10 selleck chem mM DNPH was added to the sample tube, and a volume of 200 ul of 2. 5 M HCl was added to the control tube. The tubes were incubated in the dark at 24 C for one hour. Proteins were precipitated by adding 500 ul of 20% TCA and incubating on ice for 5 min, followed by centrifugation at 10,000 g for 10 min at 4 C. The supernatant fluid was removed and the protein pellets were suspended in 1 ml of 1 1 ethanol ethyl acet ate followed by centrifugation at 10,000 g for 10 min at 4 C. Removal of supernatant fluid, suspension of pellets, and centrifugation were repeated three times. The super natant fluid was then removed and the protein pellets were dissolved in 300 ul of 6 M guanidine hydrochloride and mixed using a vortex mixer every 10 min for one hour. Ali quots of 200 ul from each tube were added to separate wells of a clear 96 well plate, and the absorbance at 370 nm was measured using a Spectra max plus 384 mi croplate reader. The corrected absorbance was calculated by subtracting the absorbance of the well containing the control tube aliquot from the absorbance of the well containing the sample tube aliquot.