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PubMed2001

Identification of human candidate genes for male infertility by digital differential display.

Evidence for the importance of genetic factors in male fertility is accumulating. In the literature and the Mendelian Cytogenetics Network database, 265 cases of infertile males with balanced reciprocal translocations have been described. The candidacy for infertility of 14 testis-expressed transcripts (TETs) were examined by comparing their chromosomal mapping position to the position of balanced reciprocal translocation breakpoints found in the 265 infertile males. The 14 TETs were selected by using digital differential display (electronic subtraction) to search for apparently testis-specific transcripts in the TIGR database. The testis specificity of the 14 TETs was further examined by reverse transcription-polymerase chain reaction (RT-PCR) on adult and fetal tissues showing that four TETs (TET1 to TET4) were testis-expressed only, six TETs (TET5 to TET10) appeared to be differentially expressed and the remaining four TETs (TET11 to TET14) were ubiquitously expressed. Interestingly, the two tesis expressed-only transcripts, TET1 and TET2, mapped to chromosomal regions where seven and six translocation breakpoints have been reported in infertile males respectively. Furthermore, one ubiquitously, but predominantly testis-expressed, transcript, TET11, mapped to 1p32-33, where 13 translocation breakpoints have been found in infertile males. Interestingly, the mouse mutation, skeletal fusions with sterility, sks, maps to the syntenic region in the mouse genome. Another transcript, TET7, was the human homologue of rat Tpx-1, which functions in the specific interaction of spermatogenic cells with Sertoli cells. TPX-1 maps to 6p21 where three cases of chromosomal breakpoints in infertile males have been reported. Finally, TET8 was a novel transcript which in the fetal stage is testis-specific, but in the adult is expressed in multiple tissues, including testis. We named this novel transcript fetal and adult testis-expressed transcript (FATE).

Chromosome Mapping↗

PubMed1993

Protein kinase C-dependent effects of 12(S)-HETE on endothelial cell vitronectin receptor and fibronectin receptor.

12(S)-HETE, a lipoxygenase metabolite of arachidonic acid induced a nondestructive and reversible endothelial cell (EC) retraction. 12(S)-HETE induced EC retraction was inhibited by protein kinase C inhibitors calphostin C and staurosporine but not by the protein kinase A inhibitor H8. The role of EC integrins alpha v beta 3 and alpha 5 beta 1 in 12(S)-HETE induced EC retraction was investigated. In confluent EC cultures, alpha v beta 3 is localized to focal adhesions at both the cell body and cell-cell borders and is colocalized with vinculin-containing focal adhesions. In contrast, alpha 5 beta 1 is primarily enriched at the cell-cell borders, demonstrating codistribution with cell cortical microfilaments and extracellular fibronectin. Both receptors were functional in mediating cell-cell or cell-matrix interactions based on the observations that specific antibodies inhibited EC adhesion to intact subendothelial matrix and disrupted the monolayer integrity. 12(S)-HETE induced a multistep, temporally defined redistribution of the alpha v beta 3-containing focal adhesions, leading to an eventual decrease in alpha v beta 3 plaques in the retracted ECs. This effect of 12(S)-HETE was inhibited by calphostin C but not by H8. The alterations of alpha v beta 3-containing focal adhesions preceded the development of EC retraction. 12(S)-HETE also enhanced EC alpha v beta 3 surface expression as revealed by immunofluorescence, flow cytometry, and digitized image analysis. 12(S)-HETE-induced alpha v beta 3 rearrangement (i.e., decreased focal adhesion localization and enhanced surface expression) did not result from altered mRNA transcription (as revealed by semi-quantitative RT-PCR analysis) or protein translation (as revealed by Western blotting). In contrast to its effect on alpha v beta 3, 12(S)-HETE did not demonstrate a temporally related, well-defined effect on the distribution pattern and the surface expression of alpha 5 beta 1, although the cell-cell border staining pattern of alpha 5 beta 1 was disrupted due to EC retraction. It is concluded that 12(S)-HETE-induced decrease of alpha v beta 3 localization to focal adhesions may contribute to the development of EC retraction and that 12(S)-HETE induced increase in alpha v beta 3 surface expression may promote adhesion of inflammatory leukocytes as well as tumor cells to endothelium.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

PubMed1993

Chromosomal bar codes produced by multicolor fluorescence in situ hybridization with multiple YAC clones and whole chromosome painting probes.

Colored chromosome staining patterns, termed chromosomal 'bar codes' (CBCs), were obtained on human chromosomes by fluorescence in situ hybridization (FISH) with pools of Alu-PCR products from YAC clones containing human DNA inserts ranging from 100 kbp to 1 Mbp. In contrast to conventional G- or R-bands, the chromosomal position, extent, individual color and relative signal intensity of each 'bar' could be modified depending on probe selection and labeling procedures. Alu-PCR amplification products were generated from 31 YAC clones which mapped to 37 different chromosome bands. For multiple color FISH, Alu-PCR amplification products from various clones were either biotinylated or labeled with digoxigenin. Probes from up to twenty YAC clones were used simultaneously to produce CBCs on selected human chromosomes. Evaluation using a cooled CCD camera and digital image analysis confirmed the high reproducibility of the bars from one metaphase spread to another. Combinatorial FISH with mixtures of whole chromosomes paint probes was applied to paint seven chromosomes simultaneously in different colors along with a set of YAC clones which map to these chromosomes. We discuss the potential to construct analytical chromosomal bar codes adapted to particular needs of cytogenetic investigations and automated image analysis.

Chromosome Banding↗

PubMed2004

Elevated expression of valosin-containing protein (p97) is associated with poor prognosis of prostate cancer.

PURPOSE: Valosin-containing protein (VCP) has been shown to be associated with metastasis and prognosis in human cancers. In the present study, the correlation of VCP with recurrence and prognosis in patients with prostate cancer (PCA) receiving conservative therapy was examined. EXPERIMENTAL DESIGN: VCP expression was analyzed immunohistochemically in 136 patients ranging from 46 to 92 years (median, 72 years), who received conservative therapy, including androgen deprivation, radiotherapy, or watchful waiting. Staining intensity of tumor cells was categorized as weaker (level 1) or equal to or stronger (level 2) than that in endothelial cells. The correlation of VCP expression between the mRNA and protein levels was examined in 10 patients. RESULTS: Thirty-two cases (23.5%) showed level 1 and 100 (76.5%) level 2 VCP expression. Quantitative reverse transcription-PCR analysis revealed greater VCPmRNA expression in level 2 (n = 5) than level 1 cases (n = 5; P < 0.05). A significant difference was observed between VCP level 1 and 2 patients in the positive rate for the digital rectal examination (P < 0.01), serum prostate-specific antigen level (P < 0.0001), cancer volume (P < 0.0001), Gleason score (P < 0.0001), stage (P < 0.0001), and progression-free and overall survival (P < 0.0001 for both). Multivariate analysis revealed VCP expression level, serum prostate-specific antigen level, and Gleason score to be independent prognosticators for progression-free and overall survival. Progression of PCA was found in 9.4% of level 1 but in 64% of level 2 patients. CONCLUSIONS: PCA with level 1 VCP expression could be treated conservatively.

Adenosine Triphosphatases↗

PubMed2026

OligoSeq: Rapid nanopore-sequencing of single-stranded oligonucleotides.

Nanopore-based DNA sequencing technology has achieved remarkable success in sequencing increasingly long DNA strands (e.g., over a million nucleotides long) for genomics research and biotechnology applications. However, the same level of progress has not been achieved for DNA oligonucleotides (usually ≤ 300 nucleotides long). Oligonucleotides play a crucial role in genome engineering efforts through oligo library generation and in DNA data storage, where they are used to encode computer information, such as binary (digital) data in DNA libraries. To enable these applications, accurate sequencing of oligonucleotides in a way that allows to assess for sequence variability, quality and length is essential. But sequencing solutions for oligonucleotides - particularly DNA primers for PCR, oligo DNA libraries used for mutagenesis or cDNA libraries used in gene expression analysis - remain inadequate. To address this gap, OligoSeq is presented as an innovative approach that integrates two complementary techniques: AmpliSeq (based on PCR) and RevSeq (based on reverse complementation with sequence-specific or random primers) to facilitate sequencing of single-stranded oligonucleotides using reference sequence anchor matches of more than ≥ 90% identity spanning from about 70% to 10% with AmpliSeq or RevSeq with random nonamers, respectively, and resolving the final reference sequence based on the most likely candidate from basecall frequencies, regardless of length and double-stranding method. OligoSeq can be integrated with nanopore sequencing technology pipelines and can be used as a reference for other sequencing platforms requiring double-stranded adapters, offering a practical and scalable alternative for standard quality control in single-stranded oligonucleotide synthesis. The use of nanopore technology, compatible with the double-stranding methods showcased, is shown to be the most cost-effective method for resolving original DNA sequences of different length and quality, and to assess its sequence variability, compared to other methods such as Illumina, PacBio or HPLC/MS.

Sequence Analysis, DNA↗

PubMed2002

Identification from public data of molecular markers of adenocarcinoma characteristic of the site of origin.

Patients presenting with metastatic adenocarcinoma of unknown origin are a common clinical problem. Their optimal management and therapy are facilitated by identification of the primary site, yet histologically these tumors are almost identical. Better tumor markers are needed to enable the assignment of metastases to likely sites of origin. In this study, hierarchical clustering of public serial analysis of gene expression data showed that adenocarcinomas and their metastases cluster according to their site of origin. A novel bioinformatic approach was developed to exploit the differences between these clusters, using diverse sources: public expression data from serial analysis of gene expression and digital differential display; and the published literature, including microarray studies. Sixty-one candidate tumor markers with expression predicted to be characteristic of the site of origin were identified. Eleven genes were tested by reverse transcription-PCR in primary adenocarcinomas from a range of sites, and seven (64%) were site-restricted. Analysis of public gene expression data sets is a powerful method for the identification of clinically relevant tumor markers.

Adenocarcinoma↗

PubMed1992

Simultaneous visualization of seven different DNA probes by in situ hybridization using combinatorial fluorescence and digital imaging microscopy.

Combinatorial labeling of probes (i.e., with two or more different reporters) increases the number of target sequences that can be detected simultaneously by fluorescence in situ hybridization. We have used an epifluorescence microscope equipped with a digital imaging camera and computer software for pseudocoloring and merging images to distinguish up to seven different probes using only three fluorochromes. Chromosome-specific centromere repeat clones and chromosome-specific "composite" probe sets were generated by PCR in which different mixtures of modified nucleotides, including fluorescein-conjugated dUTP, were incorporated. Cosmid clones were labeled similarly by nick-translation. The technique has been used to delineate the centromeres of seven different human chromosomes, on both 4',6-diamidino-2-phenylindole-stained metaphase spreads and interphase nuclei, to map six cosmid clones in a single hybridization experiment and to detect chromosome translocations by chromosome painting. Multiparameter hybridization analysis should facilitate molecular cytogenetics, probe-based pathogen diagnosis, and gene mapping studies.

Centromere↗

PubMed2004

An investigation of mammographic density and gene variants in healthy women.

This cross-sectional study examined if polymorphisms in genes that code for enzymes involved in the production and metabolism of estrogens are associated with mammographic density, a strong predictor of breast cancer risk. The study included 328 healthy women of different ethnicities who underwent mammographic screening and donated a blood or mouthwash sample for DNA analysis. After digitizing cranio-caudal views of the mammograms, we performed computer-assisted mammographic density assessment. Following DNA extraction, samples were analyzed for polymorphisms in the COMT (Val158Met), CYP1A1 (Ile462Val), CYP1B1 (Val432Leu), CYP1A2 (*1F) and CYP17 (T27C) genes using PCR-RFLP. Breast density was lower in Caucasians than in Asians. Caucasian women were less likely to carry the CYP1A1 variant allele and more likely to carry the variant alleles for CYP1B1 and COMT than women with Asian or Hawaiian ancestry. The low-activity COMT and CYP1A2 variant alleles were weakly related to lower percent mammographic density after adjustment for age, ethnicity, body mass index and reproductive variables (p for gene-dosage =0.08 and 0.05, respectively). These relations were observed in premenopausal women only and were similar in direction and magnitude after stratification by ethnicity. We found no significant associations between breast density and the variant alleles for CYP1A1, CYP1B1 and CYP17. Our data suggest lower mammographic density for women carrying the COMT and CYP1A2 variant alleles than for women carrying the common alleles, though this is the opposite of what is commonly hypothesized from the enzyme function.

Adult↗

PubMed2006

Differential expression of type X collagen in a mechanically active 3-D chondrocyte culture system: a quantitative study.

OBJECTIVE: Mechanical loading of cartilage influences chondrocyte metabolism and gene expression. The gene encoding type X collagen is expressed specifically by hypertrophic chondrocytes and up regulated during osteoarthritis. In this study we tested the hypothesis that the mechanical microenvironment resulting from higher levels of local strain in a three dimensional cell culture construct would lead to an increase in the expression of type X collagen mRNA by chondrocytes in those areas. METHODS: Hypertrophic chondrocytes were isolated from embryonic chick sterna and seeded onto rectangular Gelfoam sponges. Seeded sponges were subjected to various levels of cyclic uniaxial tensile strains at 1 Hz with the computer-controlled Bio-Stretch system. Strain distribution across the sponge was quantified by digital image analysis. After mechanical loading, sponges were cut and the end and center regions were separated according to construct strain distribution. Total RNA was extracted from the cells harvested from these regions, and real-time quantitative RT-PCR was performed to quantify mRNA levels for type X collagen and a housing-keeping gene 18S RNA. RESULTS: Chondrocytes distributed in high (9%) local strain areas produced more than two times type X collagen mRNA compared to the those under no load conditions, while chondrocytes located in low (2.5%) local strain areas had no appreciable difference in type X collagen mRNA production in comparison to non-loaded samples. Increasing local strains above 2.5%, either in the center or end regions of the sponge, resulted in increased expression of Col X mRNA by chondrocytes in that region. CONCLUSION: These findings suggest that the threshold of chondrocyte sensitivity to inducing type X collagen mRNA production is more than 2.5% local strain, and that increased local strains above the threshold results in an increase of Col X mRNA expression. Such quantitative analysis has important implications for our understanding of mechanosensitivity of cartilage and mechanical regulation of chondrocyte gene expression.

Journal Article↗

PubMed2006

Early increased MT1-MMP expression and late MMP-2 and MMP-9 activity during Angiotensin II induced aneurysm formation.

BACKGROUND: Angiotensin II (Ang II) is associated with a variety of cardiovascular diseases including aneurysm formation. The aim of this study was to evaluate the temporal changes in MT1-matrix metalloproteinase (MMP) and MMP-2 and -9 expression and activity during the course of Ang II induced experimental aneurysm formation. METHODS: Apolipoprotein E knockout mice (ApoE null) were infused with either 1000 ng/kg/min of Ang II (n = 20) or saline (n = 20) and then sacrificed at 7, 14, 21, and 28 days of infusion (n = 5/group/strain). Aortic diameters were measured by digital microscopy. Systolic blood pressure (SBP) was measured in the rodent tail. Suprarenal abdominal aortas had MT1-MMP mRNA levels and MMP-2 and MMP-9 mRNA levels and activity quantitated using reverse transcriptase-polymerase chain reaction (rt-PCR) and gelatin zymography, respectively. Statistical analyses included nonpaired t-test, Fisher's exact test, and analysis of variance (ANOVA). RESULTS: Aneurysms occurred in 40, 40, 20, and 80% of ApoE null-Ang II mice at 7, 14, 21, and 28 days, respectively. An early and significant rise in MT1-MMP mRNA occurred in ApoE null mice infused with Ang II mice, while there was no significant change in MMP-2 or MMP-9 mRNA levels. Total MMP-2 and MMP-9 activity increased over time in ApoE null mice infused with Ang II, peaking at 28 days (ANOVA, P < 0.01). SBP was significantly elevated by 7 days in ApoE null mice infused with Ang II compared to ApoE null mice infused with saline (123 +/- 16 versus 102 +/- 6 mm Hg, P < 0.05). CONCLUSIONS: Angiotensin II induces an early increase in aortic MT1-MMP expression with a subsequent increase in MMP-2 and MMP-9 activity. The process by which these changes cause aneurysm formation warrants further investigation.

Analysis of Variance↗

PubMed2002

Aquaporins in rat pancreatic interlobular ducts.

The aquaporin (AQP) family of water channels is distributed ubiquitously in many epithelia and plays a fundamental role in transmembrane water transport. The aim of this study is to identify the water transport pathway in pancreatic duct cells where most of the HCO-rich fluid originates. Using digital videomicroscopy, we measured the osmotic water permeability (P(f)) of pancreatic duct epithelium by exposing isolated rat interlobular ducts to the hypotonic solution (145 mosM). To identify mRNA and protein of AQPs expressed in duct cells, we conducted RT-PCR analysis and immunohistochemistry of the isolated duct and pancreas. The calculated P(f) (160-230 microm/s) of the isolated ducts was significantly reduced to 16-35 microm/s by 80-90% with either basolateral or luminal applications of HgCl(2). Fluid secretion evoked by secretin was almost completely abolished by a basolateral or luminal application of HgCl(2). A large amount of AQP1 and small amounts of AQP5 transcripts were detected in the isolated duct cells by RT-PCR. AQP1, but not AQP5, immunoreactivity was present in both luminal and basolateral membranes of the interlobular duct cells. Mercury-sensitive water channels are present in both luminal and basolateral membranes of rat pancreatic ducts. AQP1 of the known AQPs appears to be the main water pathway in interlobular ducts.

Animals↗

PubMed1999

Correlation of VEGF expression by leukocytes with the growth and regression of blood vessels in the rat cornea.

PURPOSE: To determine the temporal and spatial relationships between neovascularization and basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) mRNA and protein expression in the rat cornea after cautery with silver nitrate. METHODS: In female Sprague-Dawley rats, a silver nitrate applicator was placed on the central cornea to elicit circumferential angiogenesis, and blood vessel growth was quantified by digital image analysis of corneal flat-mounts. Total RNA or protein was extracted from whole corneas until 1 week after cautery, and bFGF and VEGF mRNA and protein levels were determined by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). To localize VEGF mRNA and protein, paraformaldehyde-fixed and paraffin-embedded histologic cross sections of corneas were examined by in situ hybridization and immunohistochemistry. Macrophages were identified by ED2 immunohistochemistry. To examine the regulation of VEGF, rats were treated with dexamethasone (0.5 mg/kg per day) and hyperoxia (70% O2). RESULTS: The neovascular response progresses in three phases: (1) a nonproliferative phase preceding vessel growth (< or = 48 hours after cautery); (2) a proliferative phase with maximal growth rate between 3 and 4 days; and (3) a regressive phase (day 7) with a decrease in vessel density accompanying the completion of vessel elongation. In corneas after cautery, bFGF mRNA expression was unchanged, and bFGF protein concentration decreaseed by 97% after 24 hours and returned to control levels by day 7. In contrast, VEGF164 and VEGF188 mRNA splice variants and protein peaked 48 hours after cautery, remained elevated 4 days after cautery, and decreased to near baseline by day 7. The peak concentration of VEGF in the cornea at 48 hours was calculated to be 720 pM, which is sufficient to evoke a functional response. In situ hybridization and immunohistochemistry showed VEGF expressed initially in neutrophils (24 - 48 hours) and subsequently in macrophages (4 days) adjacent to the cautery site. Treatment with either dexamethasone or systemic hyperoxia inhibited both neovascularization and the increase in VEGF expression. Dexamethasone inhibited 27% of cautery-induced VEGF upregulation at 24 hours and 23% at 48 hours, hyperoxia inhibited 32% at 24 hours and 43% at 48 hours, and combined treatment with both dexamethasone and hyperoxia had an additive effect (56% inhibition at 24 hours). CONCLUSIONS: VEGF production by leukocytes correlates temporally and spatially with cautery-induced angiogenesis in the rat cornea. Both inflammatory products and hypoxia appear to sufficiently increase VEGF expression near the cautery lesion to increase vascular permeability of limbal vessels and induce endothelial cell migration and proliferation.

Animals↗

PubMed2005

Echoguided drug infiltration in chronic prostatitis: results of a multi-centre study.

OBJECTIVES: In chronic prostatitis there are many causes that may provoke a therapeutical failure of a systemic antibiotic treatment. At the moment a consensus has not been reached on the efficacy of the many therapeutical options that are available with not one of these approaches being efficacious in all patients. In our view the main causes of treatment failure are the well-known hurdle to antibiotic diffusion inside the glandular parenchyma associated with the so-called intraprostatic bacterial biofilms and the possible presence of local auto-immune reactions. Given this background, we tested ultrasound guided intraprostate infiltration of a cocktail of antibiotics and betamethasone, for a therapeutical options. MATERIAL AND METHODS: 320 patients, referred to us because of symptoms indicative of chronic prostatitis, were enrolled in this study. The inclusion criteria were the severity of the symptoms and the failure of repeated cycles of antibiotics in the previous 12 months. At the initial consultation patients completed the NIH Prostatitis Symptoms Index (NIH-CPSI). All underwent: a) digital rectal examination (DRE), b) transrectal prostatic ultrasound scan (TRUS), c) uroflowmetry, d) cultures of first voiding and after prostatic massage urine and cultures of sperm for saprophytic and pathogen germs, yeasts and protozoa, e) DNA amplification with polymerase chain reaction (PCR) on urine and sperm, for Chlamydia trachomatis, Mycoplasmas (Ureaplasma urealyticum and Mycoplasma hominis), Gonococcus, HPV and HCV Patients on the basis of laboratory results received a cocktail of antibiotics associated with betamethasone. The cocktail was administered as prostate infiltration. Administration was repeated after 7 and 14 days. Final assessment of the efficacy of therapy included not only the NIH-CPSI scores but also the patient's subjective judgement expressed as a "percentage overall improvement". The percentage judgements were arbitrarily divided into 4 classes: 0-30% no improvement (Class I); 30-50% satisfactory improvement (Class II); 50-80% good improvement (Class III; 80-100% cured (Class IV). RESULTS: Statistical analysis of the results showed 68% of patients were included in the Class IV and 13% were no responders (Class I). CONCLUSIONS: In our opinion this is one of the more valid therapeutical approaches to chronic bacterial or abacterial prostatitis also if it requires more studies.

Adult↗

PubMed2006

ETV4 and Myeov knockdown impairs colon cancer cell line proliferation and invasion.

We have identified novel colorectal cancer-associated genes using NCBI's UNIGENE cDNA libraries. Colon cancer libraries were examined using Digital Differential Display and disease-associated genes were selected. Among these were ETV4 and MYEOV, novel colorectal cancer-associated genes. Samples of matched normal and neoplastic colon were obtained from human subjects and gene expression was quantified using real-time PCR. ETV4 gene expression was significantly increased in colonic neoplasia in comparison to matched normal colonic tissue (p<0.05). Myeov expression was also increased in colon neoplasia in comparison to matched normal tissue. The effect of siRNA-mediated knockdown of ETV4 and Myeov on cell proliferation and invasion was assessed. ETV4 knockdown resulted in a 90% decrease in cell proliferation (p<0.05) and a 67% decrease in cell invasion. Myeov knockdown resulted in a 48% decrease in cell proliferation (p<0.05) and a 36% decrease in cell invasion. These data suggest that ETV4 and Myeov may provide novel targets for therapeutic intervention.

Adenovirus E1A Proteins↗

PubMed1997

Giant cell tumor of tendon sheath is a polyclonal cellular proliferation.

Giant cell tumor of tendon sheath (GCTTS) is a common soft tissue tumor. Immunophenotypical evidence suggests it is of synovial cell origin. There is controversy regarding the underlying nature of this lesion, specifically whether it is a neoplastic or nonneoplastic (ie, reactive or hyperplastic) process. Karyotypic abnormalities have been identified in GCTTS and interpreted as evidence of neoplasia, although the finding of similar karyotypic abnormalities in unequivocally nonneoplastic proliferations raises questions about using such findings to define a neoplasm. In an attempt to resolve this uncertainty, a polymerase chain reaction (PCR)-based assay for methylation of the X-linked human androgen receptor gene (HUMARA) was used to assess whether GCTTS is a clonal or polyclonal proliferation. DNA was isolated from formalin-fixed, paraffin-embedded tissue blocks from eight cases of digital GCTTS in female subjects; two cases of hepatocellular carcinoma (HCC) were used as clonal controls. Seven of eight cases of GCTTS were informative, and each showed a polyclonal proliferation, whereas both cases of HCC were clonal. Our results indicate that GCTTS is a nonneoplastic proliferation, if one accepts that a population of cells forming a tumorous mass must show clonality to be classified as a neoplasm. Our results emphasize that simple karyotypic abnormalities do not define a neoplasm. It remains to be determined whether GCTTS is a reactive or hyperplastic process.

DNA Methylation↗

PubMed1996

High-resolution genotypic analysis of the genus Aeromonas by AFLP fingerprinting.

We investigated the ability of a recently developed genomic fingerprinting technique, named AFLP, to differentiate the 14 currently defined DNA hybridization groups (HGs) in the genus Aeromonas. We also determined the taxonomic positions of the phenospecies Aeromonas allosaccharophila, Aeromonas encheleia, Aeromonas enteropelogenes, and Aeromonas ichthiosmia, which have not been assigned to HGs yet. A total of 98 Aeromonas type and reference strains were included in this study. For the AFLP analysis, the total genomic DNA of each strain was digested with restriction endonucleases ApaI and TaqI. Subsequently, restriction fragments were selectively amplified under high-stringency PCR conditions. The amplification products were electrophoretically separated on a polyacrylamide gel and visualized by autoradiography. Following high-resolution densitometric scanning of the resulting band patterns, AFLP data were further processed for a computer-assisted comparison. A numerical analysis of the digitized fingerprints revealed 13 AFLP clusters which, in general, clearly supported the current Aeromonas taxonomy derived from DNA homology data. In addition, our results indicated that there is significant genotypic heterogeneity in Aeromonas eucrenophila (HG6), which may lead to a further subdivision of this species. A. allosaccharophila and A. encheleia did not represent a separate AFLP cluster but were found to be genotypically related to HG8/10 and HG6, respectively. In addition, the results of the AFLP analysis also confirmed the phylogenetic findings that A. enteropelogenes and A. ichthiosmia are in fact identical to Aeromonas trota (HG13) and Aeromonas veronii (HG8/10), respectively. The results of this study clearly show that the AFLP technique is a valuable new high-resolution genotypic tool for classification of Aeromonas species and also emphasize that this powerful DNA fingerprinting method is important for bacterial taxonomy in general.

Aeromonas↗

PubMed1999

Sonic hedgehog (shh) expression in developing and regenerating axolotl limbs.

Sonic hedgehog (shh) expression is detectable in the posterior mesenchyme of many developing vertebrate limbs. We have isolated an RT-PCR fragment from the axolotl, Ambystoma mexicanum, that has high identity to other vertebrate shh genes. We describe the localization of this transcript during development and regeneration and in response to tissue grafts and retinoic acid (RA) exposure in the axolotl. Even though axolotl digits show a reversed polarity of differentiation (anterior [A] to posterior [P]) when compared to other tetrapods (P to A), shh is nevertheless expressed on the posterior margin of developing and regenerating limb buds. When A cells are grafted adjacent to P cells, an ectopic domain of shh is induced. Exposure to retinoic acid (RA), a molecule known to alter pattern in all three limb axes in urodeles, results in ectopic expression of shh in anterior cells of the regeneration blastema. Prior to this induced expression in response to RA, there is an earlier response by the endogenous domain of shh, which is downregulated within the first few hours of exposure.

Ambystoma↗

PubMed2000

miniSAGE: gene expression profiling using serial analysis of gene expression from 1 microg total RNA.

The use of serial analysis of gene expression (SAGE) to determine gene expression profiles is increasing because the technique can provide absolute transcript numbers in a digital format and identify new genes. We developed a miniSAGE technique, which uses only 1 microg total RNA and reduces the amount of the starting material by 250- to 500-fold. Unlike the other modified SAGE methods, the miniSAGE technique does not require the additional PCR amplifications. The additional PCR amplifications potentially introduce bias and compromise the quantitative aspects of the SAGE method. Three key modifications in the miniSAGE technique are: (i) using the phase lock gel (PLG, Eppendorf) to increase the recovery and the purity of DNA material after each phenol extraction step; (ii) reducing the amount of linkers in the ligation, thereby minimizing their interference with SAGE ditag amplification and increasing the SAGE ditag yield; and (iii) employing the mRNA capture kit (Boehringer Mannheim) to allow the first five steps: mRNA isolation, cDNA synthesis, enzyme cleavage of cDNA, binding of the cleaved biotin-cDNA to the streptavidin-magnetic beads, ligating linkers to the bound cDNA, and the release of cDNA tags to occur within one tube to significantly reduce the loss of material between successive steps. Two fibroblast SAGE libraries have been successfully prepared. The preliminary analysis of 3838 tags from one library demonstrated a typical fibroblast gene expression pattern. This miniSAGE technique will permit a broader application of SAGE.

Biopsy↗