Nucleic Acids Res 2008, 36:3420–3435.PubMedCrossRef Authors’ contributions CC and MFA performed the experimental design, carried out the protein fractionation and electrophoresis, performed data analysis, and drafted the manuscript. DP carried out the mass spectrometry identifications. BC participated in the design of the study. EC and LC performed animal diagnosis,

collection of animal samples, isolation, molecular identification, and cultivation of mycoplasmas. SU contributed to coordination of the study and data interpretation, and helped to draft the manuscript. AA and MP conceived I-BET151 purchase the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.”
“Background Bacteriocins are bacterial peptides or proteins inhibitory to bacteria closely related to the producer. Many of the bacteriocins produced by lactic acid bacteria (LAB) have inhibitory spectra spanning beyond the genus level and have a potential in defending unwanted microflora. Since they are produced by food grade bacteria, some are being used in food preservation. However, ZD1839 manufacturer LAB bacteriocins could have a potential in

the medical field. With the increasing spread of antibiotic resistance, the need for alternative antimicrobials is growing. Most of the bacteriocins of LAB are small, heat-stable, cationic peptides and are divided into two classes; class I, the lantibiotics containing modified amino acids and class II, the non-lantibiotics having regular amino acid residues [1]. Among the regular peptide bacteriocins, those belonging to class IIa are produced by a large number of LAB and are best studied [2]. These bacteriocins have highly conserved amino acid sequences, and have a largely common inhibitory spectrum which includes pathogens like Listeria monocytogenes and Enterococcus spp. Their mode of action is different from common

antibiotics [3, 4]. Bacterial MK0683 ic50 resistance towards these bacteriocins does not appear to be common in nature [5], while in laboratory experiments Myosin resistance to some bacteriocins appear at high frequency [6, 7]. Characterization of the resistant phenotype is important for assessment of the usefulness for application of bacteriocins. The target for class IIa bacteriocins is the mannose phosphotransferase system (mpt-PTS) [8–11], and mutants lacking a bacteriocin dedicated target are insensitive to the bacteriocin. This mannose PTS is the major uptake system for mannose and glucose in the bacteria [12]. PTS components are also involved in gene regulation of catabolic operons [13]. Hence bacteriocin resistance is likely to cause multiple effects. Among the effects seen in class IIa bacteriocin resistant strains of L. monocytogenes are changes in cell envelope, alterations in fatty acid composition [14–17], and a metabolic shift [18].

2 ml 0.9% NaCl solution. The viability

of the cells was over 95% as determined by a trypan blue dye exclusion test. Then tumor tissue was cut and implanted subcutaneously to establish tumor bearing mice. Six to 10 days after implantation when subcutaneous tumor nodules reached approximately (120.5 ± 18.2) mm3, tumor model was successfully established and subjected to MK5108 cost Electric fields stimulation protocols. SPEF Exposure System SPEF generator was designed by Sun et al., Givinostat ic50 in the key laboratory of high voltage engineering and electrical new technology of Chongqing University [9]. The pulse curve was in form of unipolar exponential decay with the utmost voltage peak value 1000 V, pulse rise time ranging from 90–180 ns, pulse total duration 1–20 μs, and the frequency 1 Hz–5 kHz. Parameters in combination produced desired energy-controllable SPEF. Electric Fields Stimulation Protocols We used Tektronix TDS3032B Oscilloscope to monitor SPEF output and typical waveform captured referred to Figure 1. The parameters used for in vitro experiment referred to Table 1 : eight unipolar exponential decay pulses with each 20 μs duration (rise time was 160 ns), with amplitudes from 50 to 400 V/cm, and pulse repetition frequencies of 1, 60, 1 000, 5 000 Hz were delivered (cell exposure time was 30 minutes). Table 1 The parameters of SPEF used in SKOV3 cell suspensions. Test group Frequency (Hz) Intensity (V/cm) Rise time (ns)

Duration (μs) Stimulation

time (minutes) Group PFT�� solubility dmso Suplatast tosilate 1 1 50, 100, 150, 200, 250, 300, 350, 400 160 20 30 Group 2 60 50, 100, 150, 200, 250, 300, 350, 400 160 20 30 Group 3 1 000 50, 100, 150, 200, 250, 300, 350, 400 160 20 30 Group 4 5 000 50, 100, 150, 200, 250, 300, 350, 400 160 20 30 In the first procedure, each intensity constituted a separate experiment contained in a certain test group, and cell exposure time was 30 minutes for each intensity corresponding to a given frequency. Figure 1 Typical waveform of SPEF captured by Tektronix TDS3032B Oscilloscope. The parameters used in SKOV3 implanted tumor referred to Table 2 : eight unipolar exponential decay pulses with each 20 μs duration (rise time was 160 ns), with electric field intensity 250 V/cm, and pulse repetition frequencies of 1, 60, 1 000, 5 000 Hz were delivered (cell exposure time was 30 minutes). Table 2 The parameters of SPEF used in SKOV3 implanted tumor. Test Group Frequency (Hz) Intensity (V/cm) Rise time (ns) Duration (μs) Exposure time (minutes) test 1 1 250 160 20 30 test 2 60 250 160 20 30 test 3 1 000 250 160 20 30 test 4 5 000 250 160 20 30 In the second procedure, each frequency constituted a separate experiment, and tumor exposure time was 30 minutes for each frequency. In this paper, we adjusted, the frequency of the pulses by changing the interval between two consecutive pulses in a train, and then keeping both the duration and number of pulses constant.

The relationship between antiangiogenic therapy and metastasis remains to be determined and is an important topic for future research. Further study may provide additional drug targets, resulting in adjuvant therapies that can enhance the clinical benefits of antiangiogenic treatment. Acknowledgements We thank Jing Zhou for technical assistance. References 1. Folkman J: Tumor angiogenesis: therapeutic implications. N Engl J Med 1971, 285:1182–1186.PubMedCrossRef 2. Samaranayake

H, Määttä AM, Pikkarainen J, Ylä-Herttuala S: Future prospects and challenges of antiangiogenic cancer gene therapy. Hum Gene Ther 2010,21(4):381–96.PubMedCrossRef 3. Kerbel RS: Tumor angiogenesis. N Engl J Med 2008, 358:2039–2049.PubMedCrossRef 4. Jain RK: Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy. Science 2005, 307:58–62.PubMedCrossRef 5. Qu B, Guo L, Ma Selleckchem Caspase inhibitor J, Lv Y: Antiangiogenesis therapy might have the unintended effect of promoting tumor metastasis by increasing an alternative circulatory system. Med Hypotheses 2010,74(2):360–361.PubMedCrossRef 6. Casanovas O, Hicklin DJ, Bergers G, Hanahan D: Drug resistance by evasion of antiangiogenic targeting of VEGF signaling in late-stage HDAC cancer pancreatic islet tumors. Cancer Cell 2005, 8:299–309.PubMedCrossRef 7. Rubenstein JL, beta-catenin cancer Kim J, Ozawa T, Zhang M, Westphal

M, Deen DF, Shuman MA: Anti-

VEGF antibody treatment of glioblastoma prolongs survival but results in increased vascular cooption. Neoplasia 2000, 2:306–314.PubMedCrossRef 8. Kunkel P, Ulbricht U, Bohlen P, Brockmann MA, Fillbrandt R, Stavrou D, Westphal M, Lamszus K: Inhibition of glioma angiogenesis and growth in vivo by systemic treatment with a monoclonal antibody against vascular endothelial growth factor receptor-2. Cancer Res 2001, 61:6624–6628.PubMed 9. Cong R, Sun Q, Yang L, Gu H, Zeng Y, Wang B: Effect of Genistein on vasculogenic Phosphoglycerate kinase mimicry formation by human uveal melanoma cells. J Exp Clin Cancer Res 2009, 28:124.PubMedCrossRef 10. Miyamoto T, Min W, Lillehoj HS: Lymphocyte proliferation response during Eimeria tenella infection assessed by a new, reliable, nonradioactive colorimetric assay. Avian Dis 2002, 46:10–16.PubMedCrossRef 11. Pölcher M, Eckhardt M, Coch C, Wolfgarten M, Kübler K, Hartmann G, Kuhn W, Rudlowski C: Sorafenib in combination with carboplatin and paclitaxel as neoadjuvant chemotherapy in patients with advanced ovarian cancer. Cancer Chemother Pharmacol 2010. DOI 10. 1007/s00280–010–1276–2 12. Ebos JM, Lee CR, Cruz-Munoz W, Bjarnason GA, Christensen JG, Kerbel RS: Accelerated metastasis after short-term treatment with a potent inhibitor of tumor angiogenesis. Cancer Cell 2009, 15:232–239.PubMedCrossRef 13.

However, the cell membrane is likely to have undergone some degree of lipolysis as a result of an imbalance in calcium homeostasis [4], almost certainly from URMC-099 the exercise insult. The damage

literature often shows a high degree of inter-subject variability in CK and other cytosolic markers of EIMD, however, variability in the current study was relatively small, partly attributable to the trained status of the volunteers. The greater conditioning of these participants has almost certainly led to a repeated bout effect [31], whereby, a conditioning bout of exercise (in this case prior training) leads to a decrease in damage indices on subsequent bouts [4, 31, 32]. This is further check details supported by the low CK response seen in both groups following the exercise, when compared to the damage responses seen in untrained volunteers [19, 20]. Despite this relative learn more homogeneity, the CK response was less in the BCAA group suggesting the membrane integrity was maintained to greater extent than the placebo group. The damage response is known to be bi-phasic in nature; a primary response caused

by the mechanical stress of the exercise, followed by a secondary, transient inflammatory response over the following hours and days [4]. The subsequent inflammatory response increases protein uptake necessary for use as an energy source and/or pathways responsible for cell signaling and subsequent muscle remodeling [14, 33]. Although we cannot definitively support this postulate, it seems plausible that the greater bioavailability provided by BCAA facilitated

this response and thereby decreased secondary damage to the muscle. Our data concur with previous studies that show a peak in soreness at 48 h post-exercise [27, 32]. Furthermore, the group effects support find more previous data [20, 21, 34] showing a reduction in muscle soreness following a damaging bout of exercise with BCAA supplementation. Although the mechanism surrounding muscle soreness following a damaging bout of exercise is not well understood, it seems likely to be related to inflammation, particularly to the connective tissue elements [35] that sensitise nociceptors in muscle and hence increase sensations of pain [36]. However, previous work [20] demonstrating a reduction in soreness following BCAA supplementation also measured the acute inflammatory response (interleukin-6, a pro-inflammatory cytokine) and showed no difference between the BCAA and placebo groups. Jackman et al. [20] suggested that the increase in food or feeding per se, particularly amino acids, might be related to reductions in soreness. Although this idea is somewhat speculative and has no supporting evidence or proposed mechanism, we show similar trends in our data, but it is not possible to support or refute this theory.

citri colonization of the phyllosphere, which may be due, at least partly,

to the role of T3SS in X. citri biofilm formation. Figure 4 Analysis of T3SS gene expression in leaf-associated grown X. citri and survival of X. citri , hrp mutants and hrpB − c cells associated to leaves. (A) RT-qPCR to determine hrpG, hrpX and hrpE expression levels in X. citri grown associated to leaves. Bars indicate the expression levels of the T3SS genes at two days of leaf-associated growth relative to time 0. Values are the means of four biological replicates with three ABT-263 price technical replicates each. (B) X. citri, hrp mutants and hrpB −c strains leaf-associated survival on citrus leaves. Values represent an average of four leaves assayed for each strain. Error bars indicate the standard deviation. Proteomic analysis of statically cultured X. citri and hrpB − strains In order to gain new insights about the role of T3SS in biofilm formation, a proteomic analysis was performed to identify differentially expressed selleck chemicals proteins between X. citri and the hrpB − mutant grown statically. A total of 49 differentially expressed protein spots were detected of which 32 were up- and 17 down-regulated in the hrpB- mutant

in comparison to X. citri (Table 1). Identified proteins were used to determine enriched GO categories in biological processes and molecular function. The main enriched categories for the up- and down-regulated proteins with an average fold change of minimum ± 1.5 and p value < 0.05 BIRB 796 in the hrpB − mutant relative to X. citri were represented graphically

(Figure 5). The categories that showed a major enrichment unless in the up-regulated proteins in the hrpB- mutant include ‘metabolic process’, ‘catabolic process’, ‘biosynthetic process’ and ‘generation of precursor metabolites and energy’. Moreover, ‘cell cycle’, ‘cellular homeostasis’ and ‘cellular process’ were categories enriched in up-regulated proteins in this mutant. Most of the identified proteins in the categories of ‘transporter activity’ or ‘receptor activity’ belong to different classes of outer membrane proteins (OMPs) such as: FadL (XAC0019), that allows the passage of fatty acids [26], OmpW (XAC3664), involved in the transport of small hydrophilic molecules across the bacterial outer membrane [27] and RpfN (XAC2504), which was reported to play a role in carbohydrate transport [28]. In these categories also several TonB-dependent transporters (TBDTs), which are outer membrane transporters involved in the active uptake and/or in signal transduction [29], as well as two Oar (OmpA-related) proteins were detected as differentially expressed between the two strains. Table 1 Differentially expressed protein spots between X. citri and hrpB − strains statically cultured in XVM2 with a change abundance of minimum 1.5 fold and p value of < 0.05 (ANOVA) X. citri gene no. Protein name MOWSE score Accession no.

These results imply that the crystallite size of metallic cobalt

in the catalysts prepared from cobalt oxalate and cobalt chloride is obviously larger than that in the other two catalysts, agreeing well with the calculated results from the XRD data. The Co-N structure can be evidently detected in the catalysts synthesized from cobalt acetate, while that in the other catalysts are negligible. Therefore, the EXAFS results suggest that the Co-N bond/structure is not necessary to forming a catalytic active site toward ORR in PF-3084014 price Co-PPy-TsOH/C catalysts, while the metallic cobalt plays an important role in forming the active site. Smaller Co-Co bond distances/crystallite find more size is beneficial for enhancing the ORR performance, agreeing well with the results of Yuasa et al. [21]. In their research on Co-PPy/C catalysts, synthesized with electrochemically polymerized PPy, they found

that heat-treatment shortens the distances of Co-Co bond leading to better catalytic performance towards ORR. Figure 9 Fourier-transformed k 3 -weighted EXAFS functions at Co K-edge for Co foil and Co-PPy-TsOH/C catalysts prepared with various cobalt precursors. Conclusions Effects of cobalt precursors on electrochemical performance of Co-PPy-TsOH/C as catalyst towards Androgen Receptor Antagonist ORR have been comparatively studied, and the results have been analyzed with diverse physiochemical techniques. The following conclusions could be drawn from this research: (1) cobalt precursors affect both the catalytic activity of the Co-PPy-TsOH/C catalysts Buspirone HCl and the corresponding ORR mechanism; (2) the electrochemical performance, including both the ORR catalytic activity and the selectivity to four-electron-transfer reaction, of the Co-PPy-TsOH/C catalysts follows the order with respect to the used cobalt precursor that cobalt acetate > cobalt nitrate > cobalt chloride > cobalt oxalate;

(3) the synthesis process, especially the high-temperature pyrolysis, of the catalyst could be interfered by the used cobalt precursors, resulting in different microstructure, morphology, elemental state as well as the ORR performance; (4) lower graphitization degree of carbon and smaller crystallite/particle size of metallic cobalt and the uniform distribution in Co-PPy-TsOH/C catalysts lead to better ORR performance; (5) metallic cobalt is a main component forming the ORR active site in the Co-PPy-TsOH/C catalysts, but some other elements such as nitrogen is probably also involved; and (6) Co-N bond/structure is not necessary to forming a catalytic active site toward ORR in Co-PPy-TsOH/C catalysts, and a small-amount coexistence of CoO in the catalysts does not have an adverse effect on the electrochemical performance.

It has been demonstrated that in the LPS-neutralizing peptide, the lipid A binding motif includes a cluster of hydrophobic residues encompassed by basic aminoacids [14]. More recently, other authors underlined the pivotal role of a group of positively charged central residues with hydrophobic aminoacids distributed in the periphery [15]. The whole PCT used in our study, exhibited a plausible lipid A binding sequence between Pro82 and Pro91[14]. Also a putative lipid A binding sequence can be found between Leu101 and Val109[15] as illustrated in Figure 5. Figure 5 Putative LPS binding sites on PCT molecule. Proposed LPS binding sites include: i) 2–3

cationic aminoacids within a cluster of four (aminoacids 58–59 and aminoacids 93–95), ii) a cluster of hydrophobic residues encompassed by basic aminoacids (82–92), iii) a group of positively selleck inhibitor charged central residues with hydrophobic aminoacids in the periphery (101–109). Hydrophobic aminoacids in blue, cationic aminoacids in red and other aminoacids in orange. The LPS binding sites suggested by Japelj [14] and Bhattacharjya [15] are indicated. Close to the proposed LPS binding sites, a deep rough LPS chemical structure is showed. Flat dashed lines indicate the limits of the three post-translational processing products (N-ProCT, calcitonin and katacalcin)

of procalcitonin, while dashed forks encompass check details the peptides cleaved during post-translational processing [1, 3]. It has also been reported that the need for structural amphipathicity is probably not as an essential feature for LPS binding/neutralization as is the proximity of certain aminoacids (cationic and hydrophobic residues) within a given sequence [16]. The effects of PCT on LPS reactivity in the LAL test model suggest that PCT is equally active against both rough and smooth chemotypes.

The S. typhimurium strain SL1102 exhibits a Re chemotype LPS (deep rough) that has been previously reported as very toxic in an in vivo experimental model [17]. The E. coli 0111:B4 has a smooth chemotype endotoxin often used in studies regarding LPS binding/neutralization [18]. Therefore Silibinin PCT targets the lipid A Lazertinib ic50 portion which is a common structural feature of these LPSs. Since the molecular weight of PCT is approximately 13,000 daltons and the molecular weight of deep rough LPS is 3,000 daltons, the optimal ratio 5:1 (w/w) associated with LPS neutralization and cytokine inhibition would suggest a 1mole:1mole interaction between PCT and LPS, which could use any of the above mentioned interaction sites available on the PCT molecule. Moreover, our results provide the first evidence of the capability of PCT to significantly decrease the LPS-stimulated release of the Treg cytokine IL-10 and chemokine MCP-1 from human PBMC.

For subjects E7080 in vitro aged 65 years and above, the incidence for all fractures was 839/100,000 person-years, for non-spine fractures was 769/100,000 person-years and for hip fracture was 201/100,000 person-years. Predicted 10-year CP673451 osteoporotic fracture risk from risk factor assessment In multivariate Cox regression analysis, seven independent clinical risks factors associated with increased risk of osteoporotic fracture were identified (Table 2). Although a 10-year increase in age accounted for only a 5.8% increase in 10-year osteoporotic fracture risk, older men aged 65 years or above had a 2.7-fold higher risk of fracture compared with

younger men. Figure 1 shows the fracture risk in different age groups that was adjusted for competing risk of death along the study period. The interaction of age and other risk factors is shown in Fig. 2a. The combination of older age and history of fall was associated with a twofold increase in 10-year fracture risk after adjusting for competing death risk. Men aged 65 years or older with one or more falls per year had a 10-year risk of fracture of 31.9% compared with Ketotifen 15.8% for those younger than 65 years old. Table 2 Clinical risk factors associated with osteoporotic ON-01910 mouse fracture in Hong Kong Southern Chinese men (n = 1,810) Risk factors Subjects (%) B RR (95%

CI) P Age ≥ 65 years 1148 (63.4) 1.0 2.7 (1.2–5.8) 0.013 Age per 10 years increase   0.1 1.1 (1.0–1.1) 0.003 Grip strength <30 kg 447 (24.7) 1.2 3.3 (0.6–17.2) 0.160 History of fall within 1 year 257 (14.2) 2.7 14.5 (6.5–32.3) <0.0001 Difficulty bending forward 185 (10.2) 1.3 3.6 (1.3–9.9) 0.014 Kyphosis 78 (4.3) 1.2 3.4 (0.8–14.8) 0.100 Low back pain 510 (28.2) −0.1 0.9 (0.4–2.2) 0.895 BMI < 20 kg/m2 167 (9.2) 1.3 3.6 (1.0–12.8) 0.050 BMI per unit increase   −0.1 0.9 (0.8–0.9) <0.0001 Walking <30 min/day 167 (9.2) 0.5 1.6 (0.5–5.4) 0.457 History of fragility fracture 576 (31.8) 1.5 4.4 (2.0–9.4) <0.0001 History of clinical or morphometric spine fracture 112 (6.2) −0.3 0.7 (0.1–6.0) 0.761 History of clinical spine fracture 52 (2.9) 0.5 1.6 (0.2–12.0) 0.635 History of parental fracture 65 (3.6) −0.3 0.8 (0.1–5.7) 0.799 Use of walking aid 264 (14.6) 1.0 2.7 (1.1–6.5) 0.030 Homebound 121 (6.7) −0.5 0.6 (0.1–4.5) 0.620 Outdoor activity <60 min/day 608 (33.6) 1.4 4.1 (1.7–9.9) 0.001 Current and ever smoking 673 (37.2) 0.5 1.7 (0.8–3.5) 0.135 Current and ever drinking 43 (2.4) 1.0 2.7 (0.4–20.4) 0.326 Calcium Intake <400 mg/day 185 (10.2) 0.2 1.

Biomaterials 2007, 28:423–433.CrossRef 20. Zhang JY, Doll BA, see more Beckman EJ, Hollinger JO: Three dimensional biocompatible ascorbic acid containing scaffold for buy S3I-201 bone tissue engineering. Tissue Eng 2003, 9:1143–1157.CrossRef 21. Thamae T, Marien R, Chong L, Wu C, Baillie C: Developing and characterizing new materials based on waste plastic and agro-fibre. J Mater Sci 2008, 43:4057–4068.CrossRef 22. Wang JL, Che RS, Yang WQ, Lei JX: Biodegradable antistatic plasticizer based on citrate electrolyte doped with alkali metal salt and its poly(vinyl chloride) composites. Polym Int 2011,

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Conclusions Our data demonstrate an important role of histone modifications, including histone H3 acetylation and H3K4, H3K9 and H3K27 methylation state, in LPS-mediated IL-8 gene activation in intestinal epithelial cells. In particular we demonstrate that H3-acetyl, H3K4me2 and H3K9me2 changes are early, transient and correlate with the modulation of IL-8 transcriptional activity. Conversely, increase of H3K27me3 levels at IL-8 gene occurs later and is long lasting. Our data

provide novel insights into the epigenetic mechanisms that control transcription and gene expression in LPS response. Methods Cell culture Selleck ALK inhibitor The human colon cell lines HT-29 were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum (Life Technologies), 2 mM glutamine, penicillin (25 U/mL) and streptomycin (25 μg/mL) in a 5% CO2 atmosphere at 37°C. Cells were pretreated with Human interferon-γ (INF-γ) (Roche Applied Science, Germany) 10 ng/ml for 12 hours or control medium, washed, and then stimulated with LPS 50 ng/ml. LPS (Escherichia coli, O55:B5) were purchased from Sigma-Aldrich GW-572016 order (St. Louis, MO) and reconstituted in endotoxin-free water. 5-aza-2-deoxyazacytidine (ICN Biomedical Inc.) treatments were performed

at 5 μM and 50 μM final concentration while trichostatin (TSA) (Sigma Aldrich) was used at 25 and 100 nM. Western Blot Analysis Cell extracts were prepared in Nonidet P40 lysis buffer with 1 mM PMSF and Complete™ protease inhibitors mix (Roche, Indianapolis, IN, USA). 50 μg of proteins were resolved by electrophoresis using 10% SDS-PAGE gels and transferred to BA 85 0.45 μm PROTAN nitrocellulose filters (Schleicher & Schnell, Inc., Dassel, Germany). The blots were incubated with rabbit anti-IκB-α

antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti-γ-tubulin antibodies (Sigma-Aldrich Corp. St. Louis, MO, USA) as a control for protein loading. Immunoblots were stained with correspondent secondary antibodies IgG (Amersham Pharmacia Biotech, Buckinghamshire, UK), and revealed Clomifene with the enhanced chemiluminescence detection system IgG (ECL, Amersham Pharmacia Biotech, Buckinghamshire, UK). Western blot analyses of each sample were performed more than three times. Protein levels were quantified using the software Quantity One (Bio-Rad). Quantitative and semiquantitative RT-PCR analysis Total RNA was isolated with RNeasy extraction kit see more QIAGEN (Qiagen,GmBh) according to the manufacturer instructions. The integrity of the RNA was assessed by denaturing agarose gel electrophoresis (presence of sharp 28S, 18S and 5S bands) and spectrophotometry.