[Expression of MEK/ERK signal pathways in renal cell carcinoma with bone metastasis].

OBJECTIVE: To investigate the expression of MEK/ERK signaling pathways in renal cell carcinoma with bone metastasis, and to analyze the differences of expressions of VEGFR-2, MEK, ERK on the primary and metastasis tissue and its mechanism. METHODS: The tissue samples were obtained from 7 renal cell carcinoma patients kindly provided by Department of Urology, Peking University People's Hospital from January 1, 2009 to January 1, 2010. The expression of MEK/ERK signaling pathways was detected in the 7 renal cell carcinoma patients` primary and matched metastatic tissues with ICH, The antibody concentrations were 1:200, 1:25, and 1:250, respectively. The mutation of the twentieth exon of the PDGFRA gene, the second exon of the K-ras gene, the fifteenth exon of the Braf gene and the second exon of the MEK1 gene were detected with PCR. RESULTS: The expression intensities of VEGFR-2, MEK, and ERK were measured by H-score [intensity (1, 2, 3, or 4) multiplied by the distribution (%)]. VEGFR-2, MEK, and ERK expressions were divided into 3 groups according to the positive distribution of the tumor cells: 1, 0-5%; 2, 6%-50%; and 3, >50%, To assess intratumor heterogeneity, three distinct microscopic fields (×200) from each specimen were used to evaluate the expressions, Subsequently, the scores were averaged to obtain a single concatenated score for each tissue. VEGFR-2, MEK, and ERK expressions were assessed by 2 independent pathologists who were blinded to the clinicopathological data. The data were expressed as the mean value of the triplicate experiments. The expressions of MEK, and ERK were higher in the metastatic tissues than in the matched RCC tissues (6.10±4.10 vs. 1.33±0.51, P=0.015; 9.10±2.24 vs. 4.43± 2.84, P=0.021) while the expression of VEGFR-2 was not different between the primary and metastatic tissues (P=0.901). No mutation was detected on the twentieth exon of the PDGFRA gene, the second exon of the K-ras gene, the fifteenth exon of the Braf gene and the second exon of the MEK1 gene. CONCLUSION: MEK/ERK signaling pathways may play an important role in the metastasis and the resistance of sunitinib in RCC patients with bone metastasis.

[Knockdown of CMTM3 promotes migration and invasion of PC3 cell in vitro].

OBJECTIVE: To investigate the change of biological characteristics after stable knockdown of CKLF-like MARVEL transmembrane domain containing 3 (CMTM3) expression in PC3 by lentivirus shRNA and to reveal new therapeutic targets. METHODS: The research includes two groups: sh393 is the experimental group in which CMTM3 is knocked down in PC3 cell line; shN is the control group in which CMTM3 is negatively knocked down. The expression of CMTM3 was detected by Western blot. The migration ability of PC3 after stable knockdown was detected by Transwell and Wound healing assay. The invasion ability of PC3 was detected by Matrigel assay. RESULTS were obtained from at least three individual experiments. RESULTS: The expression of CMTM3 in sh393 group is significant lower than shN group (0.004 0±0.000 4 vs. 0.490 0±0.055 7, P<0.001) detected by Western blot. It also had statistical significance in Matrigel assays (248.6±4.5 vs. 113.0± 3.3), Transwell (203.6±1.9 vs. 103.0±1.2) and Wound healing assays (95.0±2.9 vs. 33.0±1.5) that knockdown of CMTM3 promoted migration, and invasion of PC3 cells in vitro (P<0.001). CONCLUSION: Negative correlation exists between the stable knockdown of CMTM3 and change of biological characteristics in PC3 cells, and knocking down CMTM3 affects migration, and invasion ability in PC3 cells.

[Expression of MEK/ERK signal pathways in renal cell carcinoma with bone metastasis].

OBJECTIVE: To investigate the expression of MEK/ERK signaling pathways in renal cell carcinoma with bone metastasis, and to analyze the differences of expressions of VEGFR-2, MEK, ERK on the primary and metastasis tissue and its mechanism. METHODS: The tissue samples were obtained from 7 renal cell carcinoma patients kindly provided by Department of Urology, Peking University People's Hospital from January 1, 2009 to January 1, 2010. The expression of MEK/ERK signaling pathways was detected in the 7 renal cell carcinoma patients` primary and matched metastatic tissues with ICH, The antibody concentrations were 1:200, 1:25, and 1:250, respectively. The mutation of the twentieth exon of the PDGFRA gene, the second exon of the K-ras gene, the fifteenth exon of the Braf gene and the second exon of the MEK1 gene were detected with PCR. RESULTS: The expression intensities of VEGFR-2, MEK, and ERK were measured by H-score [intensity (1, 2, 3, or 4) multiplied by the distribution (%)]. VEGFR-2, MEK, and ERK expressions were divided into 3 groups according to the positive distribution of the tumor cells: 1, 0-5%; 2, 6%-50%; and 3, >50%, To assess intratumor heterogeneity, three distinct microscopic fields (×200) from each specimen were used to evaluate the expressions, Subsequently, the scores were averaged to obtain a single concatenated score for each tissue. VEGFR-2, MEK, and ERK expressions were assessed by 2 independent pathologists who were blinded to the clinicopathological data. The data were expressed as the mean value of the triplicate experiments. The expressions of MEK, and ERK were higher in the metastatic tissues than in the matched RCC tissues (6.10±4.10 vs. 1.33±0.51, P=0.015; 9.10±2.24 vs. 4.43± 2.84, P=0.021) while the expression of VEGFR-2 was not different between the primary and metastatic tissues (P=0.901). No mutation was detected on the twentieth exon of the PDGFRA gene, the second exon of the K-ras gene, the fifteenth exon of the Braf gene and the second exon of the MEK1 gene. CONCLUSION: MEK/ERK signaling pathways may play an important role in the metastasis and the resistance of sunitinib in RCC patients with bone metastasis.

[Knockdown of CMTM3 promotes migration and invasion of PC3 cell in vitro].

OBJECTIVE: To investigate the change of biological characteristics after stable knockdown of CKLF-like MARVEL transmembrane domain containing 3 (CMTM3) expression in PC3 by lentivirus shRNA and to reveal new therapeutic targets. METHODS: The research includes two groups: sh393 is the experimental group in which CMTM3 is knocked down in PC3 cell line; shN is the control group in which CMTM3 is negatively knocked down. The expression of CMTM3 was detected by Western blot. The migration ability of PC3 after stable knockdown was detected by Transwell and Wound healing assay. The invasion ability of PC3 was detected by Matrigel assay. RESULTS were obtained from at least three individual experiments. RESULTS: The expression of CMTM3 in sh393 group is significant lower than shN group (0.004 0±0.000 4 vs. 0.490 0±0.055 7, P<0.001) detected by Western blot. It also had statistical significance in Matrigel assays (248.6±4.5 vs. 113.0± 3.3), Transwell (203.6±1.9 vs. 103.0±1.2) and Wound healing assays (95.0±2.9 vs. 33.0±1.5) that knockdown of CMTM3 promoted migration, and invasion of PC3 cells in vitro (P<0.001). CONCLUSION: Negative correlation exists between the stable knockdown of CMTM3 and change of biological characteristics in PC3 cells, and knocking down CMTM3 affects migration, and invasion ability in PC3 cells.

Rapid genomic DNA changes in allotetraploid fish hybrids.

Rapid genomic change has been demonstrated in several allopolyploid plant systems; however, few studies focused on animals. We addressed this issue using an allotetraploid lineage (4nAT) of freshwater fish originally derived from the interspecific hybridization of red crucian carp (Carassius auratus red var., ♀, 2n=100) × common carp (Cyprinus carpio L., ♂, 2n=100). We constructed a bacterial artificial chromosome (BAC) library from allotetraploid hybrids in the 20th generation (F20) and sequenced 14 BAC clones representing a total of 592.126 kb, identified 11 functional genes and estimated the guanine-cytosine content (37.10%) and the proportion of repetitive elements (17.46%). The analysis of intron evolution using nine orthologous genes across a number of selected fish species detected a gain of 39 introns and a loss of 30 introns in the 4nAT lineage. A comparative study based on seven functional genes among 4nAT, diploid F1 hybrids (2nF1) (first generation of hybrids) and their original parents revealed that both hybrid types (2nF1 and 4nAT) not only inherited genomic DNA from their parents, but also demonstrated rapid genomic DNA changes (homoeologous recombination, parental DNA fragments loss and formation of novel genes). However, 4nAT presented more genomic variations compared with their parents than 2nF1. Interestingly, novel gene fragments were found for the iqca1 gene in both hybrid types. This study provided a preliminary genomic characterization of allotetraploid F20 hybrids and revealed evolutionary and functional genomic significance of allopolyploid animals.

Adenoid reservoir for pathogenic biofilm bacteria.

Biofilms of pathogenic bacteria are present on the middle ear mucosa of children with chronic otitis media (COM) and may contribute to the persistence of pathogens and the recalcitrance of COM to antibiotic treatment. Controlled studies indicate that adenoidectomy is effective in the treatment of COM, suggesting that the adenoids may act as a reservoir for COM pathogens. To investigate the bacterial community in the adenoid, samples were obtained from 35 children undergoing adenoidectomy for chronic OM or obstructive sleep apnea. We used a novel, culture-independent molecular diagnostic methodology, followed by confocal microscopy, to investigate the in situ distribution and organization of pathogens in the adenoids to determine whether pathogenic bacteria exhibited criteria characteristic of biofilms. The Ibis T5000 Universal Biosensor System was used to interrogate the extent of the microbial diversity within adenoid biopsy specimens. Using a suite of 16 broad-range bacterial primers, we demonstrated that adenoids from both diagnostic groups were colonized with polymicrobial biofilms. Haemophilus influenzae was present in more adenoids from the COM group (P = 0.005), but there was no significant difference between the two patient groups for Streptococcus pneumoniae or Staphylococcus aureus. Fluorescence in situ hybridization, lectin binding, and the use of antibodies specific for host epithelial cells demonstrated that pathogens were aggregated, surrounded by a carbohydrate matrix, and localized on and within the epithelial cell surface, which is consistent with criteria for bacterial biofilms.

Medical treatment of craniosynostosis: recombinant Noggin inhibits coronal suture closure in the rat craniosynostosis model.

INTRODUCTION - The mechanisms underlying craniosynostosis remains unknown. However, mutations in FGFR2 are associated with craniosynostotic syndromes. We previously compared gene expression patterns of patent and synostosing coronal sutures in the nude rat and demonstrated down regulation of Noggin in synostosing sutures. Noggin expression is also suppressed by FGF2 and constitutive FGFR2 signaling [Warren et al. (2003) Nature, vol. 422, pp. 625-9; McMahon et al. (1998) Genes Dev, vol. 12, pp. 1438-52]. Thus, we therefore hypothesized that the addition of rhNoggin to prematurely fusing sutures should prevent synostosis. MATERIALS AND METHODS - Cohorts of nude rats were subjected to: 1) surgical elevation of the coronal suture (shams); 2) surgical elevation and placement of normal or FGFR2 mutant human osteoblasts onto the underlying dura (xenotransplants); or 3) xenotransplantation with co-application of heparin acrylic beads soaked with recombinant human (rh) Noggin. Eleven days post-surgery the sutures were harvested, stained, and histologically examined. RESULTS - Animals that received control osteoblasts, sham surgery, or no surgery demonstrated normal skull growth and coronal suture histology, whereas animals transplanted only with FGFR2 mutant osteoblasts showed evidence of bridging synostosis on the calvarial dural surface. Sutures treated with FGFR2 mutant osteoblasts and rhNoggin remained patent. CONCLUSION - The chimeric nude rate model is a viable model of craniosynostosis. FGFR2 mutations in osteoblasts induce bridging osteosynthesis demonstrating one of the mechanisms for premature suture fusion. Topical application of rhNoggin protein prevents craniosynostosis in the weanling nude rat xenotransplantation model of syndromic craniosynostosis.

Gene expression changes in peripheral blood mononuclear cells from multiple sclerosis patients undergoing beta-interferon therapy.

OBJECTIVE: Multiple sclerosis (MS) is a disabling idiopathic inflammatory disorder with evidence of immune dysfunction. Current therapies for MS include preparations of beta-interferon (beta IFN). We studied the gene expression patterns in peripheral blood mononuclear cells from relapsing-remitting MS patients undergoing weekly beta IFN-1a therapy (Avonex; 30 mg intramuscular) to identify biomarkers for beta IFN responsiveness. METHODS: Oligonucleotide microarrays were used for the comparative analysis of gene expression patterns from longitudinal PBMC samples taken from five patients undergoing beta IFN therapy. RESULTS: On the basis of two-fold changes in expression levels and statistical analyses we selected a candidate diagnostic set of 136 genes that were differentially expressed between pretreatment and IFN-beta-1a-treated MS patients. When we applied this gene set to cluster the specimens according to their expression profiles, the pretreatment samples clustered in one branch, and acute and chronic samples following treatment clustered in another branch. However, the chronic samples from the single clinical non-responder clustered with the pretreatment branch, suggesting that a possible reversal of beta IFN-induced gene expression may be contributing to the poor clinical response. CONCLUSIONS: These 136 genes represent potential targets for new MS therapeutics and the basis for lack of beta IFN response.

Phenotypic characterization of Streptococcus pneumoniae biofilm development.

Streptococcus pneumoniae is among the most common pathogens associated with chronic otitis media with effusion, which has been hypothesized to be a biofilm disease. S. pneumoniae has been shown to form biofilms, however, little is known about the developmental process, the architecture, and the changes that occur upon biofilm development. In the current study we made use of a continuous-culture biofilm system to characterize biofilm development of 14 different S. pneumoniae strains representing at least 10 unique serotypes. The biofilm development process was found to occur in three distinct stages, including initial attachment, cluster formation, and biofilm maturation. While all 14 pneumococcal strains displayed similar developmental stages, the mature biofilm architecture differed significantly among the serotypes tested. Overall, three biofilm architectural groups were detected based on biomass, biofilm thickness, and cluster size. The biofilm viable cell counts and total protein concentration increased steadily over the course of biofilm development, reaching approximately 8 x 10(8) cells and approximately 15 mg of protein per biofilm after 9 days of biofilm growth. Proteomic analysis confirmed the presence of distinct biofilm developmental stages by the detection of multiple phenotypes over the course of biofilm development. The biofilm development process was found to correlate not only with differential production of proteins but also with a dramatic increase in the number of detectable proteins, indicating that biofilm formation by S. pneumoniae may be a far more complex process than previously anticipated. Protein identification revealed that proteins involved in virulence, adhesion, and resistance were more abundant under biofilm growth conditions. A possible role of the identified proteins in biofilm formation is discussed.

Mapping of an autosomal dominant gene for Dupuytren's contracture to chromosome 16q in a Swedish family.

Mapping of an autosomal dominant gene for Dupuytren's contracture to chromosome 16q in a Swedish family.Dupuytren's contracture (DC) (OMIM 126900) is the most common connective tissue disease of mankind and has both heritable and sporadic forms. The inherited form is most frequently observed among the xanthochroi peoples of Northern Europe where its most common manifestations are thickening of the palmar fascia and contracture of the fingers. We ascertained a five-generation Swedish family in which DC is inherited in an autosomal dominant manner with high, but incomplete, penetrance by the end of the fifth decade. Blood was collected from all affected and informative unaffected family members for the performance of a genome-wide scan at a resolution of approximately 8 cM for all autosomes. Linkage was established to a single 6 cM region between markers D16S419 and D16S3032 on chromosome 16. A maximal two-point logarithm of odds (LOD) score of 3.18 was achieved at microsatellite marker D16S415 with four other markers in the region producing LODs of >1.5.

Century of Jackson-Weiss syndrome: further definition of clinical and radiographic findings in "lost" descendants of the original kindred.

Jackson-Weiss syndrome (JWS) is a condition consisting of craniosynostosis characterized by premature fusion of the cranial sutures and/or characteristic radiographic anomalies of the feet. The condition is inherited as an autosomal dominant trait with high penetrance and variable expressivity. Six different mutations in the fibroblast growth factor receptor 2 have been identified in patients with the clinical diagnosis of JWS. Jabs et al. [1994: Nat Genet 8:275-279] identified an Ala344Gly substitution in two branches of the family in which the clinical syndrome was originally described. This is the only publication to document this mutation in a family with the clinical diagnosis of JWS. In this study, we have identified a previously unrecognized branch of the original family with individuals that meet the clinical criteria for the diagnosis of JWS. We demonstrate that a mutation that produces the Ala344Gly substitution is present in affected members. This family illustrates the widely variable expression of the mutation, including a novel phenotype in the proband with a leg-length discrepancy and unilateral absence of the fifth digital ray in her right foot. We identify the clinical and detailed radiographic features of each affected individual and offer considerations when making the diagnosis of JWS.

Inhibition of apoptosis: a potential mechanism for syndromic craniosynostosis.

The biologic pathogenesis of syndromic craniosynostosis remains unknown. The purpose of this investigation was to determine whether specific biologic differences exist between normal calvarial osteoblasts and osteoblasts derived from patients with syndromic craniosynostosis. This study (1) examined the apoptotic rate and cell cycle of osteoblasts derived from patients with syndromic craniosynostosis, and (2) investigated for the presence of soluble factors released from syndrome-derived osteoblasts. Osteoblast cell lines were established from calvarial specimens of patients with clinically diagnosed syndromic synostosis and from normal controls. A co-culture technique was used to investigate for the presence of elaborated soluble factors. Apoptotic rate and cell cycle analyses were performed by using flow cytometry after staining with annexin V-fluorescein isothiocyanate and propidiumiodide, respectively. The apoptotic rate was significantly reduced in syndrome-derived osteoblasts as compared with control osteoblasts. Control osteoblasts co-cultured with syndromic osteoblasts demonstrated a dramatic reduction in their apoptotic rate as compared with those co-cultured with control osteoblasts. These results indicate that osteoblasts derived from patients with syndromic craniosynostosis display a lower apoptotic rate, a normal DNA synthetic rate, and the capability to reduce the apoptotic rate in normal calvarial osteoblasts through the elaboration of soluble factors.

Mapping of a gene for severe pediatric gastroesophageal reflux to chromosome 13q14.

CONTEXT: Gastroesophageal reflux (GER) has not previously been widely regarded as a hereditary disease. A few reports have suggested, however, that a genetic component may contribute to the incidence of GER, especially in its severe or chronic forms. OBJECTIVE: To identify a genetic locus that cosegregates with a severe pediatric GER phenotype in families with multiple affected members. DESIGN: A genome-wide scan of families affected by severe pediatric GER using polymorphic microsatellite markers spaced at an average of 8 centimorgans (cM), followed by haplotyping and by pairwise and multipoint linkage analyses. SETTING: General US community, with research performed in a university tertiary care hospital. SUBJECTS: Affected and unaffected family members from 5 families having multiple individuals affected by severe pediatric GER, identified through a patient support group. MAIN OUTCOME MEASURES: Determination of inheritance patterns and linkage of a genetic locus with the severe pediatric GER phenotype by logarithm-of-odds (lod) score analysis, considering a lod score of 3 or greater as evidence of linkage. RESULTS: In these families, severe pediatric GER followed an autosomal dominant hereditary pattern with high penetrance. A gene for severe pediatric GER was mapped to a 13-cM region on chromosome 13q between microsatellite markers D13S171 and D13S263. A maximum multifamily 2-point lod score of 5.58 and a maximum multifamily multipoint lod score of 7.15 were obtained for marker D13S1253 at map position 35 cM when presumptively affected persons were modeled as unknown (a maximum multipoint score of 4.88 was obtained when presumptively affected persons were modeled as unaffected). CONCLUSION: These data suggest that a gene for severe pediatric GER maps to chromosome 13q14. JAMA. 2000;284:325-334

RNA differential display of scarless wound healing in fetal rabbit indicates downregulation of a CCT chaperonin subunit and upregulation of a glycophorin-like gene transcript.

BACKGROUND/PURPOSE: Scars form as wounds heal in adult organisms. In addition to disrupting cosmetic appearance, scar tissue can cause significant morbidity, and even death if it blocks vital organ function. Previous work has established that fetal wounds, especially in early to midgestation, can heal without scarring. Because such inherent physiological mechanisms ultimately are under genetic control, a study was initiated to elucidate the differences in gene expression that produce scarless wound healing in the mammalian fetus but scarring in postnatal wounds. Reverse transcription polymerase chain reaction (RT-PCR) differential display (DD) was used to detect differentially expressed mRNA transcripts in a rabbit model of wound healing. METHODS: Adult and 21-day fetal full-thickness rabbit skin specimens from wounded and unwounded sites were harvested 12 hours postwounding. RNA extracted from the tissue was used as a template in DD reactions using anchoring and random primers to generate tissue-specific gene expression fingerprints. The over 2,000 resulting amplimers (gene transcripts) were screened for differential expression among the 4 types of specimens: fetal control (unwounded), fetal wound, adult control, and adult wound. Selected bands distinctly upregulated or downregulated in fetal wound lanes on the DD gels were excised, and the cDNA was extracted, reamplified, cloned into vectors, and sequenced. DD results were confirmed by limiting-dilution RT-PCR using sequence-specific primers. RESULTS: Differential display (DD) showed 22 amplimers that were significantly upregulated in all fetal wound samples as compared with little or no expression in fetal control, adult control, or adult wound tissues. Conversely, 5 transcripts were downregulated in the fetal wound specimens but highly expressed in the 3 comparison tissues. Reamplification of selected transcripts by PCR, followed by cloning and DNA sequencing, yielded 7 distinct sequences, each representing a gene expressed differently in fetal wound than in the other 3 tissues. A transcript that was downregulated in fetal wound showed very high sequence homology to part of the human gene for the eta subunit of the hetero-oligomeric particle CCT (the chaperonin containing T-complex polypeptide 1 or TCP-1). An upregulated amplimer showed significant DNA sequence homology to glycophorins A and B. One sequence was identified as 28S rRNA. The remaining 4 candidate sequences showed no significant homology to known genes, but 1 had high homology to expressed sequence tags of unknown function. CONCLUSIONS: With careful experimental design and proper controls and verifications, differential display of RNA expression is a potentially powerful method of finding genes that specifically regulate a particular physiological process such as fetal wound healing. No a priori knowledge of what genes might be involved, or why, is necessary. This study indicates that downregulation of a gene that codes for a chaperonin subunit and upregulation of several other genes may be involved in the striking scarless character of wound healing in the mammalian fetus. Results suggest the hypothesis that downregulation of the CCT chaperonin in fetal wound may inhibit the formation of myofibroblasts, a cell type that correlates highly with scarring in postnatal wound healing, by preventing the folding of sufficient alpha-smooth muscle actin to form the stress fibers characteristic of these cells.

Refined localization of a gene for pediatric gastroesophageal reflux makes HTR2A an unlikely candidate gene.

Our objective in this study was to determine whether mutations in the gene for the 5-hydroxytryptamine receptor 2A (HTR2A) cause the autosomal dominant form of severe pediatric gastroesophageal reflux (GER), which we had previously mapped to a 21-cM region at chromosome 13q14. Direct sequencing of the HTR2A gene was carried out on DNA from affected and unaffected members of families with severe pediatric GER displaying genetic linkage to the HTR2A locus. In addition, we performed high-resolution linkage mapping within the GER gene region using additional polymorphic markers closely linked to HTR2A. Several previously reported polymorphisms in the HTR2A gene were identified in three families affected with GER. In addition, we identified a novel polymorphism at nucleotide -1273 in the HTR2A promoter. No mutant allele cosegregated exclusively with the GER phenotype in any family. Linkage analysis using additional polymorphic markers narrowed the region of the GER gene to a 9 cM interval between markers D13S263 and CAGR1, formally excluding HTR2A as a candidate gene. In conclusion, sequence analysis of HTR2A and linkage analysis argue against mutations in HTR2A being a cause of severe pediatric GER.