The Expression of PEDF and its Putative Receptors in Hepatocellular Carcinoma and Background Liver Tissue.

BACKGROUND/AIM: Hepatocellular carcinoma (HCC) remains one of the biggest medical issues. Pigment epithelial-derived factor (PEDF) is a glycoprotein that belongs to the superfamily of serine protease inhibitors. PEDF interacts with its two receptors, adipose triglyceride lipase (ATGL) and laminin receptor (LR). MATERIALS AND METHODS: We conducted immunohistochemical staining for PEDF, LR and ATGL in 151 resected HCCs and their background liver tissues. RESULTS: High expression of LR in HCC was associated with high histological grade and portal vein invasion, while high expression of PEDF in HCC was associated with absence of portal vein invasion. High LR expression in background liver was statistically associated with low serum albumin levels and was an independent prognostic factor of worse outcomes. No cases with more than 5% fatty degeneration in the background liver tissue showed high PEDF expression. CONCLUSION: PEDF/LR/ATGL could be potential biomarkers in HCC and various chronic hepatic disorders.

The Regional Specific Alterations in BBB Permeability are Relevant to the Differential Responses of 67-kDa LR Expression in Endothelial Cells and Astrocytes Following Status Epilepticus.

Status epilepticus (a prolonged seizure activity, SE) differently affects vasogenic edema formation and dystrophin-aquaporin 4 (AQP4) expressions between the rat hippocampus and the piriform cortex (PC). In the present study, we explored whether the 67-kDa laminin receptor (LR) expression was relevant to the regional specific susceptibility of vasogenic edema at 3 days after SE. In spite of no difference in expression levels of 67-kDa LR, dystrophin, and AQP4 under physiological conditions, SE-induced serum extravasation was more severe in the PC than the hippocampus. Western blots demonstrated that SE reduced expression levels of 67-kDa LR, dystrophin, and AQP4 in the PC, but not in the hippocampus proper. Immunofluorescent studies revealed that SE increased 67-kDa LR expression in reactive CA1 astrocyte, but reduced it in the PC and the molecular layer of the dentate gyrus due to massive astroglial loss. Furthermore, SE decreased expressions of endothelial 67-kDa LR and SMI-71 (endothelial brain barrier antigen) in these regions. The 67-kDa LR neutralization evoked serum extravasation in these regions of normal animals without astroglial loss. Similar to SE, 67-kDa LR neutralization also reduced dystrophin-AQP4 expressions in the PC more than the total hippocampus. Furthermore, 67-kDa LR IgG infusion increased phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), but not c-Jun N-terminal kinase, independent of phosphoprotein enriched in astrocytes of 15 kDa (PEA15) activity. Co-treatment of U0126 (an ERK1/2 inhibitor) alleviated vasogenic edema formation and the reduced dystrophin-AQP4 expressions induced by 67-kDa LR neutralization. The 67-kDa LR IgG infusion also increased the susceptibility to SE induction. Therefore, our findings suggested that the cellular specific alterations in 67-kDa LR expression might be involved in the severity of SE-induced vasogenic edema formation in regional specific manners, which might affect the susceptibility to SE induction.

PCK3145 inhibits proliferation and induces apoptosis in breast and colon cancer cells.

AIM: To explore the effects of PCK3145 beyond prostate cancer. MATERIALS AND METHODS: Using Trypan blue, MTT proliferation assays, cell cycle and apoptosis analysis, we assessed the effects of PCK3145 on prostate (PC-3), breast (MCF-7) and colon (HT-29) human cancer cell lines and in osteosarcoma (MG-63) cells; any synergistic effects with docetaxel and oxaliplatin were also explored. RESULTS: PCK3145 inhibited proliferation and induced apoptosis of PC-3, MCF-7 and HT-29 cells in a dose- and time-dependent manner but not in the MG-63 cell line, consistent with the low expression of the laminin receptor (LR) in the latter cell line. PCK3145 produced rapid (within 5 min) and transient (up to 60 min) activation of MEK and ERK1/2. Synergistic effects were observed with docetaxel and oxaliplatin. CONCLUSION: PCK3145 can exert anticancer activity not only on prostate but also on breast and colon cancer cells, possibly through LR-mediated activation of MEK and ERK1/2 phosphorylation.

67 laminin receptor promotes the malignant potential of tumour cells up-regulating lysyl oxidase-like 2 expression in cholangiocarcinoma.

BACKGROUND: 67 laminin receptor (67LR) plays an important role in the invasion and metastasis of cholangiocarcinoma, but its mechanism remains unclear. AIMS: We investigated the clinical significance of 67LR and its relation to lysyl oxidase-like 2 (LOXL2) in 67LR-mediated invasion and metastasis in cholangiocarcinoma. METHODS: The clinical significance of 67LR and LOXL2 expression and the prognosis of patients were investigated in 73 cancerous and 32 paracancerous tissues by immunohistochemistry. The impact of LOXL2 on invasion, metastasis and 67LR expression was evaluated in cholangiocarcinoma cells by shRNA or expressed-plasmid transfection. RESULTS: Expression of 67LR was recognized in 35.62% cholangiocarcinoma tissue, and none in paracancerous tissues. LOXL2 was positively correlated with expression of 67LR. Expression of 67LR or LOXL2 in cholangiocarcinomas was significantly associated with lymph node metastasis, differentiation and poor overall survival. Cox analysis showed that 67LR can act as an independent prognostic biomarker of prognosis in cholangiocarcinoma patients. Expression of LOXL2 decreased by knockdown of 67LR and increased by overexpression of 67LR in cholangiocarcinoma cells. Knockdown of LOXL2 reduced invasion and metastasis in vitro and in vivo. CONCLUSION: 67LR may regulate the expression of LOXL2 to promote invasion and metastasis in cholangiocarcinoma cells. It could be used as an independent prognostic marker in cholangiocarcinoma patients.

Expression, purification, crystallization and preliminary crystallographic analysis of laminin-binding protein (Lmb) from Streptococcus agalactiae.

Laminin-binding protein (Lmb), a surface-exposed lipoprotein from Streptococcus agalactiae (group B streptococcus), mediates attachment to human laminin and plays a crucial role in the adhesion/invasion of eukaryotic host cells. However, the structural basis of laminin binding still remains unclear. In the context of detailed structural analysis, the lmb gene has been cloned, expressed in Escherichia coli, purified and crystallized. The crystals diffracted to a resolution of 2.5 A and belonged to the monoclinic space group P2(1), with unit-cell parameters a = 56.63, b = 70.60, c = 75.37 A, beta = 96.77 degrees .

Enhancement of gene transfer using YIGSR analog of Tat-derived peptide.

Cell penetrating peptide based gene carriers are notably known for low level of gene transfer. To remedy this, as laminin receptor (LR) has been previously linked to tumor metastasis, the LR-binding domain (YIGSR) as well as a scrambled sequence (SGIYR) were added to Tat-derived peptide sequence (YIGSR-Tat and SGIYR-Tat respectively). Peptides cellular uptake was assessed with high-LR (HT1080) and low-LR (HT29) cell lines by flow cytometry. Their ability to form complexes with DNA was examined using YOPRO-1 fluorescence assay and their transfection efficiencies evaluated using a luciferase reporter gene assay. DNA complexes were formed at (+/-) charge ratios as low as 2:1. While no conclusion could be drawn on the effect of YIGSR sequence on peptides uptake in both cell lines, a significant improvement in gene transfection in HT1080 cells was achieved using YIGSR-Tat compared to Tat and SGIYR-Tat. Additionally this increased efficiency was inhibited by excess free YIGSR. No significant difference in transfection efficiency was observed between Tat, SGIYR-Tat and YIGSR-Tat based complexes in HT29 cells. These studies demonstrate that attachment of receptor-binding ligand (YIGSR) to Tat-derived peptide can improve the efficiency of gene transfer in LR-positive cells (HT1080).

Identification of alpha-actinin 4 and 67 kDa laminin receptor as stage-specific markers in esophageal cancer via proteomic approaches.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies in the world with a very poor prognosis. The majority of ESCC patients present with advanced metastatic disease upon diagnosis. Therefore, it is important to understand the molecular mechanism in the tumor invasion process and to find new biomarkers for early diagnosis and prognostic evaluation. METHODS: Differentially expressed proteins among different stages of primary ESCCs and their matched surrounding normal tissues were compared by proteomics-based technology. The correlations between interesting proteins and clinical features of ESCC were further investigated by using ESCC tissue microarray (TMA) by immunohistochemical staining. RESULTS: Compared with normal tissues, a total of 18 differentially expressed proteins were identified in ESCC in this study. Among them, expression levels of alpha-actinin 4 (ACTN4) and 67 kDa laminin receptor (67LR) were progressively increased from stage I to III. Clinicopathological correlation using TMA revealed that overexpression of ACTN4 was significantly associated with advanced tumor stage (P = .026) and lymph node metastasis (P = .049), whereas overexpression of 67LR was significantly correlated with advanced tumor stage (P = .019) but not lymph node metastasis. CONCLUSIONS: These findings suggested that overexpression of ACTN4 and 67 LR is associated with ESCC progression and that these biomarkers may potentially be useful to prognostic evaluation, molecular biological classification, and therapeutic targeting.

67-kDa laminin receptor in human laryngeal squamous cell carcinoma.

OBJECTIVES/HYPOTHESIS: Abnormal interaction of epithelial cells with laminin component of basement membrane may account for altered biological behavior of cells, influencing proliferation, adhesion, and motility. In the current study, we investigated the role of 67-kDa laminin receptor (67LR), a high affinity receptor for laminin, in aggressiveness of laryngeal squamous cell carcinoma. METHODS: Thirty paraffin-embedded specimens and 20 fresh tissues of patients with laryngeal squamous cell carcinoma were analyzed using immunohistologic and reverse-transcriptase polymerase chain reaction techniques, respectively. Expression of 67LR on the surface of AMC-HN-8 cells was examined by flow cytometry. The effect of 67LR monoclonal antibody (MLuC5) on the adhesive and invasive abilities of AMC-HN-8 cells was determined by adherence and invasion inhibition assay in vitro. RESULTS: Both at the mRNA and protein level, laryngeal carcinoma cells expressed higher level of 67LR than normal epithelial cells (P < .01). The expression of 67LR correlated inversely with differentiation extent of tumor (P < .05). 67LR level was significantly increased in patients with lymph node metastases than those without lymph node involvement (P < .05). Flow cytometry showed 80.9 +/- 0.9% of AMC-HN-8 cells expressed 67LR. After 60 minutes and 120 minutes of incubation, MluC5 induced 57.1 +/- 3.6% and 63.2 +/- 2.8% inhibition of adhesion, respectively. The invasive ability of AMC-HN-8 cells to matrigel was reduced by MLuC5. CONCLUSIONS: Laryngeal carcinoma cells over-expressing 67LR have a stronger aggressive potential, which might make 67LR a promising target for the treatment of metastatic tumor.

Identification of oncofetal antigen/immature laminin receptor protein epitopes that activate BALB/c mouse OFA/iLRP-specific effector and regulatory T cell clones.

During tumor development in mice and humans, oncofetal Ag/immature laminin receptor (OFA/iLRP)-specific Th1, CTL, and IL-10-secreting T (Ts) cells are induced. The presence of too many Ts or too few effector T cells appears to predict a poor prognosis. We established clones of OFA/iLRP-specific splenic Th1, CTL, and Ts cells from the OFA/iLRP+ MCA1315 fibrosarcoma-bearing BALB/c mice or from BALB/c mice vaccinated with 1 or 10 microg of rOFA/iLRP. The MCA1315 tumor cell-reactive T cell clones were characterized as to surface Ag phenotype, cytokine secretion profile, and specificity for OFA/iLRP presented by syngeneic splenic APC. OFA/iLRP-specific Th1 and Ts clones were established from all mice. OFA/iLRP-specific CTL could be established from all mice except for mice immunized with 10 microg of rOFA/iLRP. Analysis of the proliferation profile of the OFA/iLRP-specific clones to overlapping OFA/iLRP 12-mer peptides that spanned the OFA/iLRP protein sequence defined the epitopes to which the T cell clones responded. There was a similar spatial distribution of the epitopes to which the two types of CD8 T cell clones responded. The nonapeptide epitopes of the Ts clones were located between aa 36 and 147 of OFA/iLRP, while the epitopes of the CTL clones were located between aa 52 and 163. Even though the CTL and Ts epitopes shared part of the protein, all of the CD8 CTL epitopes were distinct and separable from those of CD8 Ts cells.

A unique monoclonal antibody 29A stains the cytoplasm of amniotic epithelia and cutaneous basement membrane.

BACKGROUND: The basic function of epithelia is to provide a boundary between tissue and its external environment, and is achieved by a wide variety of components including extracellular molecules. Multiple monoclonal antibodies raised against epithelial antigens have helped identify a range of distinct, novel protein epitopes. OBJECT: In this study, we raised a monoclonal antibody to detect a novel epithelial molecular component. METHODS: We have produced a mouse monoclonal antibody using normal human amniotic tissue as an immunogen. The monoclonal antibody was subsequently immunohistochemically screened, and the target antigen was cloned using an immunoscreening method. RESULT: In the course of the screening, we identified unique antibody staining patterns within the cytoplasm of a subset of amniotic cells at intervals within the normal placental epithelia. By immunoscreening, we identified this candidate gene as laminin receptor (LR). By dot blot analysis, this antibody reacted with recombinant LR. The same localization of the antigen and LR was proved by a double staining immunofluorescence test in the placenta. This monoclonal antibody unexpectedly demonstrated linear staining within the dermal-epidermal junction of normal human skin but failed to react within the keratinocyte cytoplasm. CONCLUSION: We have produced and characterized a novel monoclonal antibody 29A that recognizes an LR-related molecule, which demonstrated a unique staining pattern. This monoclonal antibody might be a useful tool for further investigations into the epithelial tissues and the cutaneous basement membrane (BM).

Serotype-specific entry of dengue virus into liver cells: identification of the 37-kilodalton/67-kilodalton high-affinity laminin receptor as a dengue virus serotype 1 receptor.

Dengue virus, the causative agent of dengue fever, dengue shock syndrome, and dengue hemorrhagic fever, infects susceptible cells by initially binding to a receptor(s) located on the host cell surface. Evidence to date suggests that receptor usage may be cell and serotype specific, and this study sought to identify dengue virus serotype 1 binding proteins on the surface of liver cells, a known target organ. By using a virus overlay protein binding assay (VOPBA), in both nondenaturing and denaturing gel systems, a putative dengue virus serotype 1 binding protein of approximately 37 kDa expressed on the surface of liver (HepG2) cells was identified. Mass spectrometry analysis identified a candidate protein, the 37/67-kDa high-affinity laminin receptor. Entry of the dengue virus serotype 1 was significantly inhibited in a dose-dependent manner by both antibodies directed against the 37/67-kDa high-affinity laminin receptor and soluble laminin. No inhibition of virus entry was seen with dengue virus serotypes 2, 3, or 4, demonstrating that the 37/67-kDa high-affinity laminin receptor is a serotype-specific receptor for dengue virus entry into liver cells.

Antigens up the nose: identification of putative biomarkers for nasal tolerance induction functional studies combined with proteomics.

Intranasal autoantigen delivery is the most effective means of inducing mucosal tolerance and suppression of autoimmune disease. In an effort to identify markers of the "tolerant state", we employed proteomics technology at the level of the cervical lymph node. The analysis revealed that nasal antigen administration (without adiuvant) led to modulation of various proteins among which the most prominent were haptoglobin, nonintegrin 67 kDa laminin receptor, and MRP8. The immunoregulatory haptoglobin may qualify as (bio)marker for effective immunotherapy.

Prognostic significance of laminin, laminin receptor, and bone marrow micrometastases in breast cancer patients: are these markers of aggressive behavior and metastatic potential?

Laminin is a basement membrane glycoprotein implicated in a large number of biologic activities of cancer progression, many of which are mediated by the presence of the laminin receptor (67LR) on the cell membrane. We studied the correlations of laminin and its receptor with standardized and new prognostic factors (including bone marrow micrometastases) in a series of 112 patients with operable breast cancers. Laminin-positive cells were detected in 60% of the tumors and 67LR-positive cells in 55%; both were present in 35% of the cases. No association was found between laminin or 67LR positivity and pathologic tumor size, pathologic nodal status, grading, Ki-67, estrogen receptor status, progesterone receptor status, or bone marrow micrometastases. The only statistically significant association was with menopausal status and age, with a higher percentage of 67LR-positive tumors among premenopausal and younger patients. The median follow-up was approximately 7 years. The prognosis of disease-free survival was similar in the laminin-positive and laminin-negative subjects but was significantly better in 67LR-negative patients; there were no significant differences in overall survival. The prognostic role of laminin and 67LR in disease-free survival and overall survival varied according to nodal status. In the absence of nodal involvement, the risk of relapse (and death) was greater in the patients who were positive for laminin, 67LR, or both than in those who were negative for laminin, 67LR, or both; in the case of 4 or more involved nodes, the prognostic role of laminin and 67LR was reversed. These results did not change after adjustment for age, menopausal status, tumor status, nodal status, grading, or bone marrow micrometastases.

Col IV and Fn distribution in prostatic adenocarcinoma and correlation of 67LR, MMP-9 and TIMP-1 expression with Gleason score.

OBJECTIVE: To assess the immunoreactivity of 5 proteins related to basement membrane (BM) and extracellular matrix in order to investigate whether any of them correlates with differentiation of prostatic adenocarcinoma (PAc). Two of these markers are collagen type IV (Col IV), the collagenous component of basement membrane, and fibronectin (Fn), an adhesion protein in extracellular matrix. Others are matrix metalloproteinase-9 (MMP-9), a type IV collagenase, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), which has a high affinity for MMP-9, and 67-kd laminin receptor (67LR), which belongs to the non-integrin laminin binding receptor family. STUDY DESIGN: Forty-three PAc cases with Gleason scores ranging between 5 and 10 and 10 benign prostatic hyperplasia (BPH) cases, the control group, were included in the study. Formalin-fixed and paraffin-embedded tissue slides from each case were immunostained with the avidin-biotin-peroxidase method. Immunoreactivity was determined by means of a scoring system similar to the Gleason scoring system. RESULTS: Overexpression of Col IV, Fn, 67LR and MMP-9 was detected in PAc as compared with BPH, whereas no difference was determined in TIMP-1 expression. Among these, only 67LR correlated statistically with Gleason score. CONCLUSION: Expression of 67LR in tumor cells was significantly increased in parallel to tumor grade. This may be useful in microscopic evaluation of PAc.

Interaction of integrin receptors with extracellular matrix is involved in trophoblast giant cell migration in bovine placentomes.

Integrins are heterodimeric glycoproteins involved in cell-cell and cell-extracellular matrix adhesion and signal transduction. We evaluated the distribution and the putative role of integrin receptors and extracellular matrix (ECM) proteins during trophoblast giant cell (TGC) migration and fusion with uterine epithelial cells in the cow. Placentomes from 24 cows, covering day 80 to day 270 of gestation, were used for indirect immunohistochemistry against integrin subunits alpha(1), alpha(2), alpha(3), alpha(4), alpha(5), alpha(6), alpha(v), beta(1), beta(3), beta(4)and ECM proteins collagen type I and IV, fibronectin, laminin. The basement membranes of fetal and maternal epithelia and endothelia were immunoreactive for laminin, fibronectin and collagen IV. Collagens I and IV were found in maternal stroma, while fibronectin was present in fetal and maternal stroma. The integrin subunits alpha(2), alpha(6)and beta(1)were observed in basal aspects of fetal and maternal epithelial and endothelial cells. Additionally, the alpha(6)and beta(1)integrin subunits were colocalized with laminin on TGC. The integrin alpha(2)subunit was also found on TGC, but localized with a strong gradient to the basal side. Cells of the maternal connective tissue, including endothelium, expressed alpha(1), alpha(2), alpha(3), alpha(5), alpha(6), alpha(v), beta(3)and beta(4). The expression of alpha(2), alpha(5), alpha(v), beta(3)and beta(4) occurred mainly in the septal tips. Cells of the fetal mesenchyme were positive for integrin subunits alpha(1), alpha(2), alpha(3), alpha(4), alpha(5), alpha(6), and beta(1). Our results indicate that alpha(2)beta(1)collagen and alpha(6)beta(1)laminin receptors anchor epi- and endothelial cells to basement membranes. We suggest that TGC migrate along a matrix of laminin and maintain cell-cell contact with mononuclear trophoblast cells via alpha(2)beta(1)heterodimers. Integrins in maternal stroma and fetal mesenchyme may be involved in the regulation of proliferation and differentiation of maternal septa and fetal villi.

[The expression of acetyl-heparanase, laminin and laminin receptor and their role in the metastasis of ovarian cancer].

AIM: To explore the expressions of acetyl-heparanase mRNA, laminin ( LN) and laminin receptor ( LR) in 50 ovarian carcinoma, 33 ovarian carcinoma with lymph node metastasis, and 10 serous ovarian cystadenoma as well as their role in the metastasis of ovarian cancer. METHODS: The transcription level of acetyl-heparanase mRNA, expressions of LN and LR were detected by in situ hibridization and immunohistochemical staining, respectively. RESULTS: The transcription level of acetyl-heparanase mRNA in ovarian carcinoma tissue and metastatic lymph nodes increased significantly, but its expression in primary focus was notably higher than that in metastatic lymph nodes (P < 0. 05 ). There was low expression of acetyl-heparanase mRNA in serous ovarian cystadenoma. The expression of acetyl-heparanase mRNA in malignant and benign tumor tissues had markedly difference (P < 0. 01 ). Expressions LN in both tissues mentioned above decreased while LR expression was high. The expression of acetyl-heparanase mRNA was negative correlation with that of LN, while positive with that of LR. CONCLUSION: The correlation among expressions of acetyl-heparanase mRNA, LN and LR suggests that heparanase is involved in the growth, invasion and metastasis of ovarian carcinoma.

[Experimental study of multidrug resistance mediated by human laminin receptor in gastric cancer cells].

OBJECTIVE: To study the mechanisms of multidrug resistance (MDR) mediated by human 67 000 laminin receptor (LR) with a relative molecular mas of 67 000 in gastric cancer cells. METHODS: Antisense RNA expression vector corresponding to LR precursor (LRP) was constructed by DNA recombinant technique, and transferred into gastric cancer MDR cells SGC7901/VCR with Lipofect AMINE. Western blot was employed to determine the LR expression level in gastric cancer cells. The sensitivity of gastric cancer cells to chemotherapeutic drugs was evaluated with MTT assay. Flow cytometry was used to analyze the cell cycle and to assess the mean fluorescence intensity of intracellular adriamycin in gastric cancer cells. RESULTS: Western blotting analysis demonstrated a decreased expression level of LR in SGC7901/VCR cells transfected with LRP antisense RNA expression vector. In comparison with the gastric cancer cells with out transfection or transfected with invalid vector, LR down-regulated transfectants (SGC7901/VCR-anLRP) showed higher sensitivity to vincristine, adriamycin, 5-fluodrouracil and cisplatin, and increased accumulation and retention of adriamycin. Cell cycle analysis suggested G1 block and spontaneous apoptosis of SGC7901/VCR-anLRP cells. CONCLUSION: LR might take part in mediation of MDR in gastric cancer cells through interfering with drug accumulation and cell apoptosis.

Detection of the 67-kD laminin receptor in prostate cancer biopsies as a predictor of recurrence after radical prostatectomy.

OBJECTIVES: Reliable prognostic indicators are needed for a better pretherapeutic assessment of the agressiveness of organ-confined prostate cancer (PC) lesions. The 67-kD laminin receptor (67LR) is a cell-surface-associated protein involved in the acquisition of the invasive and metastatic phenotype of a variety of human cancer cell types. We have previously shown that 67LR detection in PC tissues from radical prostatectomy (RP) specimens is an independent predictor of biochemical (PSA) relapse in patients with clinically localized PC. In this study, we assessed 67LR detection in diagnostic PC biopsies as a predictor of biochemical relapse after RP. METHODS: Diagnostic biopsy and subsequent RP tissue specimens from 151 patients with clinically localized PC were immunohistochemically analyzed for 67LR expression. The level of 67LR expression was evaluated by both intensity and extent of the staining. Clinicopathological preoperative and postoperative parameters, including 67LR expression, were correlated with each other and tested as predictors of biochemical relapse. RESULTS: 67LR was detected in 67.5 and 68.2% of biopsies and RPs, respectively. 67LR detection in RP specimens was an independent predictor of relapse. The level of 67LR expression in the biopsy was significantly associated with the biopsy Gleason score (p<0.05) but failed to predict the pathological stage (p>0.1). Biochemical progression-free estimates for patients whose biopsy did or did not express the protein differed with only borderline statistical significance (p = 0.05). Multivariate analysis identified biopsy Gleason score as the only independent preoperative predictor of recurrence. Significant discrepancies in levels of 67LR expression were found between matched biopsy and RP specimens (p<0.05), with exact agreement rates <40%. CONCLUSIONS: 67LR detection in PC biopsies was not a significant preoperative predictor of outcome after RP. Heterogeneity of 67LR expression and biopsy sampling errors most likely represented the main reasons for discordant results between biopsy and RP specimens.

Use of western immunoblot for evaluation of myocardial dystrophin, alpha-sarcoglycan, and beta-dystroglycan in dogs with idiopathic dilated cardiomyopathy.

OBJECTIVE: To evaluate the potential importance of dystrophin, alpha-sarcoglycan (adhalin), and beta-dystroglycan, by use of western blot analysis, in several breeds of dogs with dilated cardiomyopathy. SAMPLE POPULATION: Myocardial samples obtained from 12 dogs were evaluated, including tissues from 7 dogs affected with dilated cardiomyopathy, 4 control dogs with no identifiable heart disease (positive control), and 1 dog affected with Duchenne muscular dystrophy (negative control for dystrophin). Of the affected dogs, 4 breeds were represented (Doberman Pinscher, Dalmatian, Bullmastiff, and Irish Wolfhound). PROCEDURE: Western blot analysis was used for evaluation of myocardial samples obtained from dogs with and without dilated cardiomyopathy for the presence of dystrophin and 2 of its associated glycoproteins, alpha-sarcoglycan and beta-dystroglycan. RESULTS: Detectable differences were not identified between dogs with and without myocardial disease in any of the proteins evaluated. CONCLUSIONS AND CLINICAL RELEVANCE: Abnormalities in dystrophin, alpha-sarcoglycan, and beta-dystroglycan proteins were not associated with the development of dilated cardiomyopathy in the dogs evaluated in this study. In humans, the development of molecular biological techniques has allowed for the identification of specific causes of dilated cardiomyopathy that were once considered to be idiopathic. The use of similar techniques in veterinary medicine may aid in the identification of the cause of idiopathic dilated cardiomyopathy in dogs, and may offer new avenues for therapeutic intervention.

Anomalous dystroglycan in carcinoma cell lines.

Dystroglycan is a receptor responsible for crucial interactions between extracellular matrix and cytoplasmic space. We provide the first evidence that dystroglycan is truncated. In HC11 normal murine and the 184B5 non-tumorigenic mammary human cell lines, the expected beta-dystroglycan 43 kDa band was found but human breast T47D, BT549, MCF7, colon HT29, HCT116, SW620, prostate DU145 and cervical HeLa cancer cells expressed an anomalous approximately 31 kDa beta-dystroglycan band. alpha-Dystroglycan was udetectable in most of the cell lines in which beta-dystroglycan was found as a approximately 31 kDa species. An anomalous approximately 31 kDa beta-dystroglycan band was also observed in N-methyl-N-nitrosurea-induced primary rat mammary tumours. Reverse transcriptase polymerase chain reaction experiments confirmed the absence of alternative splicing events and/or expression of eventual dystroglycan isoforms. Using protein extraction procedures at low- and high-ionic strength, we demonstrated that both the 43 kDa and approximately 31 kDa beta-dystroglycan bands harbour their transmembrane segment.