Low-abundance mRNAs Research Articles

Osmotic Swelling Induces p75 Neurotrophin Receptor (p75NTR) Expression via Nitric Oxide

Brain injuries by physical trauma, epileptic seizures, or microbial infection upset the osmotic homeostasis resulting in cell swelling (cerebral edema), inflammation, and apoptosis. Expression of the neurotrophin receptor p75NTR is increased in the injured tissue and axon regeneration is repressed by the Nogo receptor using p75NTR as the signal transducer. Hence, p75NTR seems central to the injury response and we wished to determine the signals that regulate its expression. Here, we demonstrate that tonicity mediated cell swelling rapidly activates transcription of the endogenous p75NTR gene and of a p75NTR promoter-reporter gene in various cell types. Transcription activation is independent of de novo protein synthesis and requires the activities of phospholipase C, protein kinase C, and nitric-oxide synthase. Hence, p75NTR is a nitric oxide effector gene regulated by osmotic swelling, thereby providing a strategy for therapeutic intervention to modulate p75NTR functions following injury.

Lagepläne

Pluripotent mouse P19 embryonal carcinoma (EC) cells have been extensively used as a developmental model system because they can differentiate in the presence of retinoic acid (RA) into derivatives of all three germ layers depending on RA dosage and culture conditions. The expression of several genes has been shown to be induced in RA-treated P19 EC cells and, interestingly, some of these genes may play important roles during mouse embryogenesis. In view of the increasing evidence that RA is a crucial signaling molecule during vertebrate development, we have initiated a study aimed at the systematic isolation of genes whose expression is induced in P19 cells at various times after exposure to RA. We describe here an efficient differential subtractive hybridization cloning strategy which was used to identify additional RA-responsive genes in P19 cells. Fifty different cDNA fragments corresponding to RA-induced genes were isolated. Ten cDNAs represent known genes, 4 of which have already been described as RA-inducible, while the remaining 40 correspond to novel genes. Many of these cDNA sequences represent low-abundance mRNAs. Kinetic analysis of mRNA accumulation following RA treatment allowed us to characterize four classes of RA-responsive genes. We also report the sequence and expression pattern in mouse embryos and adult tissues of one of these novel RA-inducible genes, Stral, and show that it corresponds to the mouse ligand for the Cek5 receptor protein-tyrosine kinase.

Low editing efficiency of GluR2 mRNA is associated with a low relative abundance of ADAR2 mRNA in white matter of normal human brain.

The ionotropic glutamate receptor (GluR) subunits GluR2, GluR5 and GluR6 are subject to RNA editing at their Q/R sites, resulting in significant alterations in the channel properties of the receptors. RNA editing at the Q/R site of GluRs is both developmentally and regionally regulated. Here we provide the first quantitative measurements of both mRNAs of the GluR subunits and mRNAs of the RNA editing enzymes ADAR1-ADAR3 in a comparison of the efficiency of editing at the Q/R site with the expression levels of ADAR mRNA in human brain. We demonstrate that the Q/R site of GluRs in white matter is edited significantly less than in grey matter. In addition, by means of quantitative reverse transcription-polymerase chain reaction methods, we demonstrate that the relative abundance of ADAR2 mRNA to GluR2 mRNA is significantly lower in white matter than in grey matter and that the GluR2 Q/R site editing decreased only when the ratio of ADAR2 mRNA (not that of ADAR1 mRNA) to GluR2 mRNA dropped below a threshold (20 x 10(-3)). These results suggest that Q/R site of GluRs editing is regulated in a regional, and hence presumably cell-specific, manner and that the GluR2 Q/R site editing is critically regulated by ADAR2 in human brain.

Optimization of RNA yield, purity and mRNA copy number by treatment of urine cell pellets with RNA later

Background: We have shown that measurement of mRNA for cytotoxic attack proteins perforin and granzyme B in urinary cells is a noninvasive means of diagnosing acute rejection of human renal allografts. Urinary cell mRNA studies have yielded useful information in other patient populations such as patients with cancer. The isolation of sufficient and high quality ribonucleic acid (RNA) from urinary cells however is problematic. RNA later, an RNA stabilization solution, has been reported to optimize RNA isolation from tumor tissues stored at room temperature and from pigment-rich ocular tissues. Methods: We explored whether the addition of RNA later to urine cell pellets improves RNA yield, enhances purity and facilitates measurement of low abundance mRNAs. We measured, with the use of real-time quantitative polymerase chain reaction (PCR) assay, levels of expression of a constitutively expressed gene 18S rRNA and mRNA for granzyme B and transforming growth factor-β 1 (TGF-β 1) in urine specimens and renal biopsies obtained from renal allograft recipients. Results: RNA yield ( P<0.01, Wilcoxon signed rank test) and the A260/ A280 ratio ( P<0.01) were both higher with urine cell pellets treated with RNA later prior to snap freezing compared to cell pellets that were not treated with RNA later prior to snap freezing. Levels (copy number per 1 μg of total RNA) of 18S rRNA ( P<0.02), granzyme B mRNA ( P=0.002) and TGF-β 1 ( P=0.02) were all higher with treated urine cell pellets compared to untreated cell pellets. Kruskall–Wallis one way analysis of variance and pair-wise comparisons with Student–Newman–Keuls test showed that the levels of mRNA for granzyme B ( P<0.05) and TGF-β 1 ( P<0.05) are significantly different between renal allograft biopsies and untreated urine cell pellets but not between the biopsy specimens and RNA later-treated urine cell pellets. Conclusions: The addition of RNA later to urine cell pellets improves RNA isolation from urinary cells and facilitates measurement of low abundance mRNAs.

Quantitation of mRNA levels of steroid 5α-reductase isozymes: a novel method that combines quantitative RT-PCR and capillary electrophoresis

A novel, accurate, rapid and modestly labor-intensive method has been developed to quantitate specific mRNA species by reverse transcription-polymerase chain reaction (RT-PCR). This strategy combines the high degree of specificity of competitive PCR with the sensitivity of laser-induced fluorescence capillary electrophoresis (LIF-CE). The specific target mRNA and a mimic DNA fragment, used as an internal standard (IS), were co-amplified in a single reaction in which the same primers are used. The amount of mRNA was then quantitated by extrapolation from the standard curve generated with the internal standard. PCR primers were designed to amplify both a 185 bp fragment of the target cDNA for steroid 5α-reductase 1 (5α-R1) and a 192 bp fragment of the target cDNA for steroid 5α-reductase type 2 (5α-R2). The 5′ forward primers were end-labeled with 6-carboxy-fluorescein (6-FAM). Two synthetic internal standard DNAs of 300 bp were synthesized from the sequence of plasmid pEGFP-C1. The ratio of fluorescence intensity between amplified products of the target cDNA (185 or 192 bp fragments) and the competitive DNA (300 bp fragment) was determined quantitatively after separation by capillary electrophoresis and fluorescence analysis. The accurate quantitation of low-abundance mRNAs by the present method allows low-level gene expression to be characterized.

The gastrointestinal tract as target of steroid hormone action: Quantification of steroid receptor mRNA expression (AR, ERα, ERβ and PR) in 10 bovine gastrointestinal tract compartments by kinetic RT-PCR

We have examined the tissue-specific mRNA expression pattern of androgen receptor (AR), both estrogen receptor (ER) subtypes ERα and ERβ and progestin receptor (PR) in 10 bovine gastrointestinal compartments. Goal of this study was to evaluate the deviating tissue sensitivities and the influence of the estrogenic active preparation Ralgro ® on the compartment-specific expression regulation. Ralgro ® contains Zeranol which shows strong estrogenic and anabolic effects. Eight heifers were treated for 8 weeks with Ralgro ® at different dosages (0, 1, 3, and 10 times). To quantify the very low abundant steroid receptor mRNA transcripts sensitive and reliable real-time (kinetic) reverse transcription (RT)-PCR quantification methods were validated on the LightCycler. Expression results indicate the existence of AR and both ER subtypes in all 10 gastrointestinal compartments. PR receptor was expressed at very low abundancy. Gastrointestinal tissues exhibit a specific ERα and ERβ expression pattern with high expression levels for both subtypes in rectum, colon and ileum. With increasing Zeranol concentrations a significant down-regulation for ERα and ERβ was observed in jejunum ( P<0.001 and <0.05, respectively). Significant up-regulations under estrogen treatment could be shown in abomasum for ERα ( P<0.05) and in rectum for ERβ ( P<0.001). The authors conclude, that especially estrogens and the expression of their corresponding receptor subtypes may play an important role in the modulation and regulation in gastric as well as gut functions, cell proliferation and possibly in the pathophysiology of cell cancer. The different expression patterns of ERα and ERβ can be regarded as support of the hypothesis that the subtype proteins may have different biological functions in the gastrointestinal tract. AR and PR seem to be not estrogen dependent.

Non-radioactive in situ detection of mRNA in ES cell-derived cardiomyocytes and in the developing heart.

Non-radioactive in situ hybridisation is an excellent method to visualise mRNA molecules within their topographical context. Recently we have reported a new non-radioactive in situ hybridisation procedure on tissue sections that is essentially based on the whole mount in situ hybridisation procedure. This method is superior in spatial resolution and sensitivity compared to the radioactive in situ hybridisation procedure. Generally, low levels of gene expression, such as found with the developmental onset of gene expression and in differentiating embryonic stem cells, are difficult to detect by in situ hybridisation. Here an application of the protocol is presented which is based on tyramide signal amplification, which enables the detection of very low abundant mRNAs. The significance of this method is two-fold: (1) the molecular phenotype of embryonic stem cell-derived cardiomyocytes can be examined at the cellular level with high sensitivity, and (2) the number of cells that express the gene of interest can be assessed.

Sensitivity and accuracy of quantitative real-time polymerase chain reaction using SYBR green I depends on cDNA synthesis conditions

The recent development of real-time PCR has offered the opportunity of sensitive and accurate quantification of mRNA levels that is crucial in biomedical research. Although reverse transcription (RT)-PCR is at present the most sensitive method available, many low abundant mRNAs are, although detectable, often not quantifiable. Here we report an improved two-step real-time RT-PCR procedure using SYBR green I and the LightCycler that better permits accurate quantification of mRNAs. Omission of dithiothreitol from the cDNA synthesis reaction was found to be crucial. This resulted in a lower cycle number at which the cDNA level is determined ( C T value), steeper amplification curves, and removal of background fluorescence in the subsequent PCR. In addition, the choice of the cDNA priming oligo can improve detection sensitivity even further. In contrast to hexamer primer usage, both gene-specific and oligo-dT VN priming were very efficient and accurate, with gene-specific priming being the most sensitive. Finally, accurate quantification of mRNAs by real-time PCR using SYBR green I requires verification of the specificity of PCR by both melting curve and gel analysis.

Detection of oligonucleotide hybridization at femtomolar level and sequence-specific gene analysis of the Arabidopsis thaliana leaf extract with an ultrasensitive surface plasmon resonance spectrometer.

A flow-injection (FI) device is combined, through the use of a low-volume (4 microl) flow cell, with an ultrasensitive surface plasmon resonance (SPR) spectrometer equipped with a bi-cell photodiode detector. The application of this novel FI-SPR device for sequence-specific ultratrace analysis of oligodeoxynucleotides (ODNs) and polydeoxynucleotides was demonstrated. Self-assembled monolayers of ODN probes are tethered onto Au films with a mercaptohexyl group at the 3' ends. The FI-SPR provides a detection level (< or =54 fM) 2-3 orders of magnitude lower than other SPR devices and compares well with several ultrasensitive detection methods for labeled DNA targets (e.g. fluorophore-tagged and radiolabeled DNA samples). The technique is also highly selective, since a 47mer ODN target with a single-base mismatch yielded a much smaller SPR signal, and a specific interaction was detected when the complementary target was present at 0.001% of the total DNA. The FI-SPR was extended to the measurement of two individual genes in a cDNA mixture transcribed from an Arabidopsis thaliana leaf mRNA pool. The greatly enhanced sensitivity not only obviates the necessity of DNA labeling, but also significantly reduces sample consumption, allowing direct quantification of low abundance mRNAs in cellular samples without amplification.

Unprocessed proinsulin promotes cell survival during neurulation in the chick embryo.

We have chosen a vertebrate model accessible during neurulation, the chick, for analysis of endogenous insulin signaling and its contribution to early embryonic cell survival. Unlike rodents, humans and chickens have a single preproinsulin gene, facilitating its prepancreatic expression characterization. We show that in vivo interference with embryonic insulin signaling using antisense oligonucleotides against the insulin receptor increases apoptosis during neurulation. In contrast, high glucose administration does not increase the level of apoptosis in culture or in vivo. Exogenous insulin and, remarkably, proinsulin achieve similar survival protective effects at 10(-8) mol/l. The low abundant preproinsulin mRNA from the prepancreatic embryo is translated to a protein that remains as unprocessed proinsulin. This concurs with the absence of prohormone convertase 2 (PC2) in the embryo, whereas PC2 is present later in embryonic pancreas. A C-peptide--specific antibody stains proinsulin-containing neuroepithelial cells of the chick embryo in early neurulation, as well as other cells in mesoderm- and endoderm-derived structures in the 2.5-day embryo. We have determined by 5'-RACE (rapid amplification of cDNA ends), and confirmed by RNase protection assay, that prepancreatic and pancreatic proinsulin mRNA differ in their first exon, suggesting differential transcriptional regulation. All these data support the role of endogenous proinsulin in cell survival in the chick embryo during important pathophysiologic periods of early development.

A New Quantitative Method of Real Time Reverse Transcription Polymerase Chain Reaction Assay Based on Simulation of Polymerase Chain Reaction Kinetics

Real-time reverse transcription (RT) PCR is currently the most sensitive method for the detection of low-abundance mRNAs. Two relative quantitative methods have been adopted: the standard curve method and the comparative CT method. The latter is used when the amplification efficiency of a reference gene is equal to that of the target gene; otherwise the standard curve method is applied. Based on the simulation of kinetic process of real-time PCR, we have developed a new method for quantitation and normalization of gene transcripts. In our method, the amplification efficiency for each individual reaction is calculated from the kinetic curve, and the initial amount of gene transcript is derived and normalized. Simulation demonstrated that our method is more accurate than the comparative CT method and would save more time than the relative standard curve method. We have used the new method to quantify gene expression levels of nine two-pore potassium channels. The relative levels of gene expression revealed by our quantitative method were broadly consistent with those estimated by routine RT-PCR, but the results also showed that amplification efficiencies varied from gene to gene and from sample to sample. Our method provides a simple and accurate approach to quantifying gene expression level with the advantages that neither construction of standard curve nor validation experiments are needed.

Accurate quantitative RT-PCR for relative expression of Slo splice variants

Much interest has been shown in the use of multi-template reverse transcription-polymerase chain reaction (RT-PCR) as a quantitative instrument for low-abundance mRNAs. A desire to achieve finely-graded quantification of the stress- and hormone-related regulation of one splicing decision in an ion channel gene motivated us to test the reliability of simultaneous amplification of two splice variants with one pair of flanking constitutive primers. Unexpectedly indiscriminate heteroduplexing between the two amplification products, despite a large length difference, and their tight comigration with one homoduplex, mandated a rigorously-denaturing electrophoresis protocol. Conveniently, a new fluorescent dye with high affinity for single-stranded DNA has become available. Though the dye has a good dynamic range, we found that dye and gel saturation compounded by the length difference between products introduced an asymmetrical error into the calculation of relative abundance. Avoiding several pitfalls, dye calibration could be used to correct the error. We also found that differences in the amplification efficiency of the two templates were not constant, but dependent on the initial template ratio, requiring a non-linear correction. Together these improvements gave us very consistent quantitative results, and thus advance our analysis of hormonal mechanisms underlying the regulation of alternative splicing of an ion channel critically involved in stress responses.

Regulation of Mitochondrial sn-Glycerol-3-phosphate Acyltransferase Activity: Response to Feeding Status Is Unique in Various Rat Tissues and Is Discordant with Protein Expression

Triacylglycerol plays a critical role in an organism's ability to withstand fuel deprivation, and dysregulation of triacylglycerol synthesis is important in the development of diseases such as obesity and diabetes. Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and committed step of glycerolipid synthesis and is therefore a potential site for regulation of triacylglycerol synthesis. Because several studies suggest that triacylglycerol synthesis is linked to the mitochondrial isoform, we studied mitochondrial GPAT expression and the effect of feeding status on the regulation of mitochondrial GPAT in various rat tissues. Liver, adipose, and soleus muscle have high levels of GPAT mRNA, but low protein expression, whereas heart and adrenal, tissues with low GPAT mRNA abundance, have the highest GPAT protein expression. In addition, heart, which has the highest expression of mitochondrial GPAT protein, has low mitochondrial GPAT specific activity (0.02 nmol/min/mg). Liver and adipose have the highest mitochondrial GPAT specific activity (0.17 nmol/min/mg), but very low protein expression. Discrepancies between GPAT protein expression and activity suggest that mitochondrial GPAT may be regulated acutely. In response to a 48-h fast, liver and adipose mitochondrial GPAT protein expression and activity decrease 30–50%. After 24-h refeeding of either chow or high-sucrose diet, mitochondrial GPAT protein expression and activity overshoot normal levels 30–60%. In kidney, mitochondrial GPAT protein and activity increase 65 and 30%, respectively, with refeeding, whereas in the heart, mitochondrial GPAT activity increases 2.3-fold after a fast, with no change in protein expression. We also found that hepatic mitochondrial GPAT activity in the neonatal rat constitutes a lower percentage of the total GPAT activity than in the adult. We postulate that GPAT expression is modulated uniquely in each tissue according to specific needs for triacylglycerol storage.

A sensitive technique to clone low abundance receptor transcripts from single microdissected tissue punches

Tissue microdissection is a rapidly growing technique with wide applicability in the field of gene expression analysis as improved RNA extraction and reverse-transcription polymerase chain reaction (RT-PCR) techniques provide the sensitivity to amplify transcription products from increasingly small numbers of cells. In spite of these advances, isolation, cloning and regional localization of rare or low-abundance mRNA from very small tissue samples remain a difficult and challenging task, especially when high degenerate primers are to be used. We have addressed this problem using a combination of optimized techniques and purification steps added between individual reaction steps. The extreme sensitivity resulting from these modifications permits cloning of new members of a closely homologous gene family from only one microdissected tissue sample and widens the applicability of tissue microdissection. Using this protocol, nested degenerate PCR primers were designed to amplify members of the large and relatively homologous olfactory receptor (OR) gene family from RNA extracted from 125-μm diameter punches of tissue microdissected from 16-μm sections of the main olfactory bulb (MOB) of the mouse. Levels of OR mRNA in these punches are extremely low, due to the small volume of tissue and the low abundance of OR mRNA in MOB tissue. Several ORs were amplified, cloned and sequenced from a series of individual tissue punches, and in situ hybridization was used to verify the presence of mRNA corresponding to the cloned OR sequences in MOB sections.

Direct in situ reverse transcriptase-polymerase chain reaction.

In situ hybridization has been used for localization of specific nucleic acid sequences at the cellular level despite providing relatively low-detection sensitivity. In situ reverse transcriptase-polymerase chain reactions (RT-PCR) enhance sensitivity and thus enable localization of low-abundance mRNA in a cell. However, the available methods are fraught with problems of nonspecific amplifications as a result of mispriming and/or amplification from partially digested residual genomic DNA in tissue. Herein, we demonstrate that nonspecific background amplification can be eliminated by pretreatment of samples with restriction enzymes before DNase I digestion. Primers tagged with a far-red shifted fluorescent dye such as Cy5 in PCR reactions allow identification of target mRNA by fluorescence microscopy. These novel modifications lead to increased specificity and rapid in situ detection of cellular mRNA and thus may be used for pathological diagnosis.

RNA fingerprinting of specific plant cell types: Adaptation to plants and optimization of RNA arbitrarily primed PCR (RAP-PCR)

Differential gene expression contributes to cell differentiation and underlies plant responses to hormonal and environmental factors. Most methods available to identify differentially expressed genes in plants are biased towards moderately or strongly expressed genes. RNA arbitrarily primed polymerase chain reaction (RAP-PCR) produces populations of amplicons from reverse transcribed RNA in a process similar to differential display, but with a higher degree of reproducibility and sensitivity, thus enabling the identification of low abundance mRNA. A detailed RAP-PCR protocol allowing the rapid identification of differentially expressed genes in scarce plant cells, such as stomatal guard cells, is presented here. In addition, a fast and reliable method for the semi-quantitative confirmation of gene expression patterns is described.

Improved non-radioactive Northern blot protocol for detecting low abundance mRNAs from mammalian tissues

A rapid and sensitive non-radioactive Northern blot protocol is described which has been optimised in several critical steps. This is based on a formaldehyde-denaturing agarose gel electrophoresis, an alkaline transfer, hybridisation with digoxigenin-DNA probes and detection with a chemiluminescent substrate. This method allows even low abundance mRNAs to be detected in total RNA samples from mammalian tissues.

Sensitive nonradioactive detection of mRNA in tissue sections: novel application of the whole-mount in situ hybridization protocol.

The relative insensitivity of nonradioactive mRNA detection in tissue sections compared to the sensitive nonradioactive detection of single-copy DNA sequences in chromosome spreads, or of mRNA sequences in whole-mount samples, has remained a puzzling issue. Because of the biological significance of sensitive in situ mRNA detection in conjunction with high spatial resolution, we developed a nonradioactive in situ hybridization (ISH) protocol for detection of mRNA sequences in sections. The procedure is essentially based on the whole-mount ISH procedure and is at least equally sensitive. Increase of the hybridization temperature to 70C while maintaining stringency of hybridization by adaptation of the salt concentration significantly improved the sensitivity and made the procedure more sensitive than the conventional radioactive procedure. Thicker sections, which were no improvement using conventional radioactive ISH protocols, further enhanced signal. Higher hybridization temperatures apparently permit better tissue penetration of the probe. Application of this highly reliable protocol permitted the identification and localization of the cells in the developing heart that express low-abundance mRNAs of different members of the Iroquois homeobox gene family that are supposedly involved in cardiac patterning. The radioactive ISH procedure scarcely permitted detection of these sequences, underscoring the value of this novel method.

Fluorescence in situ hybridization of scarce leptin receptor mRNA using the enzyme-labeled fluorescent substrate method and tyramide signal amplification.

To increase the sensitivity of fluorescence in situ hybridization (FISH) for detection of low-abundance mRNAs, we performed FISH on cryostat sections of rat hypothalamus with biotin-labeled riboprobes to leptin receptor (ObRb) and amplified the signal by combining tyramide signal amplification (TSA) and Enzyme-Labeled Fluorescent alkaline phosphatase substrate (ELF) methods. First, TSA amplification was done with biotinylated tyramide. Second, streptavidin-alkaline phosphatase was followed by the ELF substrate, producing a bright green fluorescent reaction product. FISH signal for ObRb was undetectable when TSA or ELF methods were used alone, but intense ELF FISH signal was visible in hypothalamic neurons when the ELF protocol was preceded by TSA. The TSA-ELF was combined with FISH for pro-opiomelanocortin (POMC) and neuropeptide Y (NPY) mRNAs by hybridizing brain sections in a cocktail containing digoxigenin-labeled riboprobes to NPY or POMC mRNA and biotin-labeled riboprobes to ObRb mRNA. Dioxigenin-labeled NPY or POMC mRNA hybrids were subsequently detected first with IgG-Cy3. Then biotin-labeled leptin receptor hybrids were detected with the TSA-ELF method. Combining the ELF and TSA amplification techniques enabled FISH detection of scarce leptin receptor mRNAs and permitted the identification of leptin receptor mRNA in cells that also express NPY and POMC gene products.

Galectin-1 and Galectin-3 in Chronic Pancreatitis

Galectin-1 and galectin-3 have important functions in cell-cell interactions, cell adhesion to extracellular matrix, the organization of extracellular matrix, and tissue remodeling. To assess their potential role in chronic pancreatitis (CP), we examined their expression by Northern blot analysis, in situ hybridization, immunohistochemistry, and Western blot analysis in normal and CP pancreatic tissues. Northern blot analysis revealed a 4.5-fold increase of galectin-1 mRNA (p < 0.01) and a 3.8-fold increase of galectin-3 mRNA (p < 0.01) in CP samples compared with normal controls. In situ hybridization analysis of normal pancreas indicated low abundance of galectin-1 mRNA in fibroblasts, whereas galectin-3 mRNA was moderately present in ductal cells. CP samples exhibited moderate to intense galectin-1 mRNA signals in fibroblasts, whereas galectin-3 mRNA signals were intense in the cells of ductular complexes and weak in the degenerating acinar cells. In addition, intense galectin-1 and galectin-3 mRNA signals were present in nerves of normal and CP samples. Immunohistochemistry showed a distribution pattern of galectin-1 and galectin-3 similar to that described for in situ hybridization. Relative quantification of galectin-1 and galectin-3 protein by immunoblotting revealed an increase of 3.2-fold and 3.0-fold, respectively, in CP compared with normal controls. There was a significant correlation between galectin-1 and fibrosis and between galectin-3 and fibrosis and the density of ductular complexes. Up-regulation of galectin-1 in fibroblasts and galectin-3 in ductular complexes suggests a role of these lectins in tissue remodeling in CP. Galectin-1 might participate in ECM changes, whereas galectin-3 seems to be involved in both ECM changes and ductular complex formation.