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PubMed1995

Linkage analysis with multiplexed short tandem repeat polymorphisms using infrared fluorescence and M13 tailed primers.

The use of short tandem repeat polymorphisms (STRPs) as marker loci for linkage analysis is becoming increasingly important due to their large numbers in the human genome and their high degree of polymorphism. Fluorescence-based detection of the STRP pattern with an automated DNA sequencer has improved the efficiency of this technique by eliminating the need for radioactivity and producing a digitized autoradiogram-like image that can be used for computer analysis. In an effort to simplify the procedure and to reduce the cost of fluorescence STRP analysis, we have developed a technique known as multiplexing STRPs with tailed primers (MSTP) using primers that have a 19-bp extension, identical to the sequence of an M13 sequencing primer, on the 5' end of the forward primer in conjunction with multiplexing several primer pairs in a single polymerase chain reaction (PCR) amplification. The banding pattern is detected with the addition of the M13 primer-dye conjugate as the sole primer conjugated to the fluorescent dye, eliminating the need for direct conjugation of the infrared fluorescent dye to the STRP primers. The use of MSTP for linkage analysis greatly reduces the number of PCR reactions. Up to five primer pairs can be multiplexed together in the same reaction. At present, a set of 148 STRP markers spaced at an average genetic distance of 28 cM throughout the autosomal genome can be analyzed in 37 sets of multiplexed amplification reactions. We have automated the analysis of these patterns for linkage using software that both detects the STRP banding pattern and determines their sizes. This information can then be exported in a user-defined format from a database manager for linkage analysis.

Chromosome Mapping↗
PubMed2004

Thirtyfold multiplex genotyping of the p53 gene using solid phase capturable dideoxynucleotides and mass spectrometry.

A mass spectrometry (MS) based multiplex genotyping method using solid phase capturable (SPC) dideoxynucleotides and single base extension (SBE), named the SPC-SBE, has been developed for mutation detection. We report here the simultaneous genotyping of 30 potential point mutation sites in exons 5, 7, and 8 of the human p53 gene in one tube using the SPC-SBE method. The 30 mutation sites, including the most frequently mutated p53 codons, were chosen to explore the high multiplexing scope of the SPC-SBE method. Thirty primers specific to each potential mutation site were designed to yield SBE products with sufficient mass differences. This was achieved by tuning the mass of some primers using modified nucleotides. Genomic DNA was amplified by multiplex PCR to produce amplicons of the three p53 exons. The 30 primers were combined with the PCR products and biotinylated dideoxynucleotides for SBE to generate 3'-biotinylated extension DNA products. These products were then captured by streptavidin-coated magnetic beads, while the unextended primers and other components in the reaction were washed away. The pure extension DNA products were subsequently released from the solid phase and analyzed with MS. We simultaneously genotyped 30 potential mutation sites in the p53 gene from Wilms' tumor, head and neck tumor, and colorectal tumor. Both homozygous and heterozygous genotypes were accurately determined with digital resolution. This is the highest level of multiplex genotyping reported thus far using MS, indicating that the approach might be applicable to screening a repertoire of genotypes in candidate genes as potential disease markers.

Base Sequence↗
PubMed2007

Assessment of the presence of mucosal human papillomaviruses in malignant melanomas using combined fluorescent in situ hybridization and chemiluminescent immunohistochemistry.

BACKGROUND: The vast majority of studies aimed at detecting human papillomavirus (HPV) DNA in skin cancer have used sensitive polymerase chain reaction (PCR) methods but the PCR technique, despite its high sensitivity, is not suitable to ascertain whether (i) the presence of HPV can be related only to few cells harbouring the virus, (ii) the presence of HPV is due to a tumour surface contamination and (iii) the presence of HPV is localized in cancer cells, rather than in normal keratinocytes present in the tumour biopsy. In a recent work we have found mucosal high-risk (HR) HPV genotypes in primary melanoma by PCR. OBJECTIVES: To localize mucosal HR-HPV nucleic acids and tumoural melanocytic marker in the same sections of primary melanoma samples in order to understand the relationship between HPVs and melanoma cells. METHODS: We have developed a very sensitive method that combines an enzyme-amplified fluorescent in situ hybridization (ISH) for the detection of HPV nucleic acids (types 16 and 18) with a chemiluminescent immunohistochemistry (IHC) method for the detection of the tumoural melanocytic marker HMB-45 sequentially in the same section. Digital images of fluorescent ISH and chemiluminescent IHC were separately recorded, assigned different colours and merged using specific software for image analysis. RESULTS: The combined fluorescent ISH and chemiluminescent IHC demonstrated a sharp colocalization (in the range 60-80%) of HPV nucleic acids and melanoma marker inside the same sections of melanoma biopsies, with a strong specificity and sensitivity. CONCLUSIONS: The strong colocalization of mucosal HR-HPV nucleic acids and HMB-45 melanocytic marker emphasized that viral nucleic acids were specifically present in melanoma cells and supported a possible active role of HPV in malignant melanoma.

Adult↗
PubMed2003

The implication of assessing a polymorphism in estrogen receptor alpha gene in the risk assessment of osteoporosis using a screening tool for osteoporosis in Asians.

Both genetic and environmental factors interact to determine bone mass and the risk for developing postmenopausal osteoporosis. Recently, an Asian-specific tool, the Osteoporosis Self-Assessment Tool for Asians (OSTA), has been developed to assess the risk of osteoporosis in women. An index is calculated by multiplying the difference in body weight in kilograms and age in years by 0.2 and disregarding the decimal digits. The risk of osteoporosis is classified as high, intermediate or low according to the OSTA index less than -4, -4 to -1 and greater than -1. In the present study we examined how a single nucleotide polymorphism (SNP) in exon 8 of the estrogen receptor alpha (ERalpha) gene affected the predictive value of the OSTA index. Subjects consisted of 358 postmenopausal women who were at least 55 years old. BMDs were measured by DXA, and the SNP in the ERalpha gene was assessed by PCR-RFLP. When considering both the OSTA index and ERalpha genotype in a logistic regression model, it was found that both the OSTA index and the ERalpha genotype independently contributed to the risk of osteoporosis. The odds ratios were 1.58 (95% CI 1.26-1.91) and 2.51 (95% CI 1.42-4.44) for one unit decrement in the OSTA index and each copy of the A allele of the ERalpha genotype, respectively. The joint effect conformed more to a multiplicative model of interaction than an additive model. This suggests that persons with the high-risk genotype are at far greater risk of developing osteoporosis with advancing age or decreasing body weight, the two variables from which the OSTA index is derived. Targeting preventive measures for osteoporosis subjects with risk factors and also disease-susceptibility alleles is likely to be more cost effective.

Aged↗
PubMed2005

Nocturnal changes in leaf growth of Populus deltoides are controlled by cytoplasmic growth.

Growing leaves do not expand at a constant rate but exhibit pronounced diel growth rhythms. However, the mechanisms giving rise to distinct diel growth dynamics in different species are still largely unknown. As a first step towards identifying genes controlling rate and timing of leaf growth, we analysed the transcriptomes of rapidly expanding and fully expanded leaves of Populus deltoides Bartr. ex. Marsh at points of high and low expansion at night. Tissues with well defined temporal growth rates were harvested using an online growth-monitoring system based on a digital image sequence processing method developed for quantitative mapping of dicot leaf growth. Unlike plants studied previously, leaf growth in P. deltoides was characterised by lack of a base-tip gradient across the lamina, and by maximal and minimal growth at dusk and dawn, respectively. Microarray analysis revealed that the nocturnal decline in growth coincided with a concerted down-regulation of ribosomal protein genes, indicating deceleration of cytoplasmic growth. In a subsequent time-course experiment, Northern blotting and real-time RT-PCR confirmed that the ribosomal protein gene RPL12 and a cell-cycle gene H2B were down-regulated after midnight following a decrease in cellular carbohydrate concentrations. Thus, we propose that the spatio-temporal growth pattern in leaves of P. deltoides primarily arises from cytoplasmic growth whose activity increases from afternoon to midnight and thereafter decreases in this species.

Carbohydrate Metabolism↗
PubMed2002

Erythropoietin modulates calcium influx through TRPC2.

Mammalian isoforms of calcium-permeable Drosophila transient receptor potential channels (TRPC) are involved in the sustained phase of calcium entry in nonexcitable cells. Erythropoietin (Epo) stimulates a rise in intracellular calcium ([Ca](i)) via activation of voltage-independent calcium channel(s) in erythroid cells. Here, involvement of murine orthologs of classical TRPC in the Epo-modulated increase in [Ca](i) was examined. RT-PCR of TRPC 1-6 revealed high expression of only TRPC2 in Epo-dependent cell lines HCD-57 and Ba/F3 Epo-R, in which Epo stimulates a rise in [Ca](i). Using RT-PCR, Western blotting, and immunolocalization, expression of the longest isoform of mTRPC2, clone 14, was demonstrated in HCD-57 cells, Ba/F3 Epo-R cells, and primary murine erythroblasts. To determine whether erythropoietin is capable of modulating calcium influx through TRPC2, CHO cells were cotransfected with Epo-R subcloned into pTracer-CMV and either murine TRPC2 clone 14 or TRPC6, a negative control, into pQBI50. Successful transfection of Epo-R was verified in single cells by detection of green fluorescent protein from pTracer-CMV using digital video imaging, and successful transfection of TRPC was confirmed by detection of blue fluorescent protein fused through a flexible linker to TRPC. [Ca](i) changes were simultaneously monitored in cells loaded with Rhod-2 or Fura Red. Epo stimulation of CHO cells cotransfected with Epo-R and TRPC2 resulted in a rise in [Ca](i) above base line (372 +/- 71%), which was significantly greater (p < or = 0.0007) than that seen in cells transfected with TRPC6 or empty pQBI50 vector. This rise in [Ca](i) required Epo and extracellular calcium. These results identify a calcium-permeable channel, TRPC2, in erythroid cells and demonstrate modulation of calcium influx through this channel by erythropoietin.

Animals↗
PubMed2001

[A case of bilateral coronal craniosynostosis with the P250R mutation in FGFR3 gene].

Recently, the substitution of proline 250 by arginine in the fibroblast growth factor receptor 3 (FGFR3) gene, has been identified in patients with craniosynostosis and defines a new syndrome on a molecular basis. We report a 1-year-1-month-old female with bilateral coronal craniosynostosis who had the P250R mutation in FGFR3 gene detected by DNA sequencing. She had brachycephaly, temporal bossing, high and flat forehead, hypertelorism, mild proptosis, low set ears and no digital abnormalities. She underwent surgical repair at 7 months and her cosmetic problems were improved. Her development was normal up to 13 months of age. DNA analysis from her parents showed that her father had the same mutation. The phenotypes of the P250R mutation in the FGFR3 syndrome are variable even within the same family, but main characteristic clinical features are follows, 1) lateral or bilateral coronal craniosynostosis, 2) mild hand and foot anomalies, and 3) sensory deafness. In FGFR3 syndrome the diagnosis of P250R mutation by polymerase chain reaction (PCR) is very easy and important for early diagnosis and genetic counseling.

Abnormalities, Multiple↗
PubMed2000

Tissue gene expression analysis using arrayed normalized cDNA libraries.

We have used oligonucleotide-fingerprinting data on 60,000 cDNA clones from two different mouse embryonic stages to establish a normalized cDNA clone set. The normalized set of 5,376 clones represents different clusters and therefore, in almost all cases, different genes. The inserts of the cDNA clones were amplified by PCR and spotted on glass slides. The resulting arrays were hybridized with mRNA probes prepared from six different adult mouse tissues. Expression profiles were analyzed by hierarchical clustering techniques. We have chosen radioactive detection because it combines robustness with sensitivity and allows the comparison of multiple normalized experiments. Sensitive detection combined with highly effective clustering algorithms allowed the identification of tissue-specific expression profiles and the detection of genes specifically expressed in the tissues investigated. The obtained results are publicly available (http://www.rzpd.de) and can be used by other researchers as a digital expression reference.

Algorithms↗
PubMed2002

[Identification of digital differential expression patterns of a novel human gene (UBAP1) by an expressed sequence tag strategy].

BACKGROUND & OBJECTIVES: This study was designed to identify differential expression patterns in tumor and normal tissues of the human novel gene, UBAP1, a putative nasopharyngeal carcinoma (NPC) relate gene, which is located at human chromosome 9p21-22 where loss of heterozygosity frequently occurs in NPC. METHODS: Based on the generation of expressed sequence tags (ESTs), in the authors' electronic Northern blot, complete cDNA of ubap1 gene was taken as a "probe" sequence, and human EST Database search of GenBank was carried out using BLASTn programs. A report generated in a BLAST search of ubap1 gene sequence against an EST database consisting of such 'pedigree' ESTs allows the inference of the gene's tissue distribution. Furthermore, differential RT-PCR was used to describe the expression patterns of ubap1 in partial tumor tissues. RESULTS: Database surveys indicated ubap1 gene was not only ubiquitously expressed in many normal tissues with various levels but also differentially expressed in different tumor tissues, especially down-regulated in multiple neoplastic tissues such as brain, breast, skin, colon, testis, and uterus tumor tissues. Furthermore, differential RT-PCR analysis demonstrated expression of ubap1 was down-regulated or absent in 7 of 11(64%) meningioma samples and 13 of 18 (72%) colorectal tumor tissues respectively. CONCLUSIONS: The authors present an EST approach that proved to be useful for the identification of differential expression patterns of ubap1 in different tumors. These findings could be valuable for the investigation of the mechanism of UBAP1 gene exhibiting differential expression, potentially involved in the progression of multiple cancers.

Carrier Proteins↗
PubMed2000

Expression of parathyroid hormone-related peptide (PTH-rp) and its receptorin the porcine ovary: regulation by transforming growth factor-beta and possible paracrine effects of granulosa cell PTH-rp secretion on theca cells.

Parathyroid hormone-related peptide (PTH-rp) and the PTH-rp receptor are expressed in certain cancers as well as in many normal tissues. To evaluate the expression of this Ca(2+)-regulating hormone and its receptor in porcine ovary, we isolated partial cDNAs encoding homologous PTH-rp and PTH-rp receptor using reverse transcription-polymerase chain reaction (RT-PCR). The cDNA encoding PTH-rp (419 base pairs [bp]) was 92% and 87% homologous to human and rat sequences, respectively, while the PTH-rp receptor clone (167 bp) was 94% and 91% identical to the human and rat genes. Qualitative estimates of PTH-rp mRNA by RT-PCR indicated that the PTH-rp gene is expressed at high levels in the corpus luteum but is undetectable in granulosa and theca cells isolated from small (1-5 mm) and medium-sized (5-8 mm) antral follicles. In contrast, PTH-rp receptor transcripts were most abundant in corpora lutea and theca cells, and least abundant (albeit detectable) in granulosa cells. Regulation of PTH-rp protein production was assessed in serum-free monolayer cultures of porcine granulosa cells. Transforming growth factor (TGF)-beta1 (100 ng/ml) increased PTH-rp concentrations (assayed by two-site immunoradiometric assay of culture media) as well as corresponding PTH-rp mRNA accumulation (assessed by RT-PCR) in a time-dependent manner, with maximal responses of 3- to 5-fold at 96 h. TGF-beta1 dose-response studies revealed an ED(50) of 0. 24-0.38 ng/ml with a maximal effect at 30 ng/ml. Other growth factors and hormones, including insulin, insulin-like growth factor (type I), epidermal growth factor, FSH, estradiol, and interleukin-1, failed to alter PTH-rp secretion. Biological effects of PTH-rp were evident in purified porcine theca cells. Using the Ca(2+)-sensitive fluorescent indicator dye, fura-2, and digital imaging videomicroscopy, we found that PTH-rp (1 microM) stimulated intracellular free calcium ion concentrations ([Ca(2+)](i)) in single porcine theca cells. The [Ca(2+)](i) elevation was characterized by a slow and prolonged rise. After PTH-rp stimulation, theca cells maintained responsiveness to hormone stimulation by LH, which elicited a typical theca cell [Ca(2+)](i) response. Our results allow a hypothesis of a paracrine intrafollicular signaling system involving interaction between theca cell-derived TGF-beta and granulosa cell-derived PTH-rp, with feedback by PTH-rp on theca cells. Alternatively, expression of mRNAs encoding PTH-rp and its receptor in corpora lutea suggests that this peptide may play a role in luteal cell function. The precise role of this intraovarian PTH-rp system will require further study.

Animals↗
PubMed2001

Terminal restriction fragment patterns: a tool for comparing microbial communities and assessing community dynamics.

Terminal Restriction Fragment (TRF) patterns, also known as Terminal Restriction Fragment Length Polymorphisms (T-RFLP), are a recently introduced PCR-based tool for studying microbial community structure and dynamics. Since the first review of TRF methodology (Marsh, 1999. Curr. Op. Microbiol. 2: 323-7), at least 35 new research articles were published that include this powerful tool in some part of their reports. This review covers some of the applications that TRF patterns were used for and provides a discussion of how to create and analyze TRF pattern data. This data has the advantage of being simply and rapidly produced using standard DNA sequencing equipment. The raw data are automatically converted to a digitized form that can be easily analyzed with a variety of multivariate statistical techniques. The identification of specific elements in a TRF pattern is possible by comparison to entries in a good sequence database or by comparison to a clone library. As an added advantage when investigating complex microbial communities such as those in soils and intestines, TRF patterns are recognized as having better resolution than other DNA-based methods for evaluating community structure.

Animals↗
PubMed2003

Bacterial diversity in maize rhizospheres: conclusions on the use of genetic profiles based on PCR-amplified partial small subunit rRNA genes in ecological studies.

A cultivation-independent approach based on polymerase chain reaction (PCR)-amplified partial small subunit rRNA genes and genetic profiling by single-strand conformation polymorphism (SSCP) was used to characterize the bacterial diversity inhabiting the rhizosphere of maize plants grown on an agricultural field. The community structures of two cultivars, a genetically engineered and a nonengineered variety, different herbicide regimes and soil tillage were compared with each other at two sampling dates. SSCP-profiles were generated with DNA from bacterial cell consortia with primers hybridizing to evolutionarily highly conserved rRNA gene regions. On silver-stained gels, each profile consisted of approx. 50 distinguishable bands. Similarity analyses of patterns recorded by digital image analyses could not detect any difference between cultivars or treatments that was greater than the variability between replicates. A total of 54 sequences recovered from different bands were identified and grouped into operational taxonomical units (OTUs). Surprisingly, only five of 40 OTUs contained sequences of both samplings. Three different bands from a profile were selected to test whether this small overlap was due to an incomplete recovery of sequences. From a faint band, two different OTUs were found when 12 clones were analysed, and from two strong bands 24 and 22 OTUs were detected from a total of 26 and 36 clones, respectively. The OTUs belonged to phylogenetically different groups of bacteria. Gene probes that were developed to target different bands of the profiles, however, indicated in Southern blot analyses that patterns between treatments, replicates and samplings, and even from two different growing seasons were highly conserved. Our study demonstrates that community profiles can consist of more sequences than detectable by staining and that gene probes in Southern blot can be a useful control to investigate the composition of microbial communities by genetic profiles.

Bacteria↗
PubMed1997

Functional analysis by imaging of melanophore pigment dispersion of chimeric receptors constructed by recombinant polymerase chain reaction.

Analysis of functional aspects of the molecular structure of proteins often requires a means to selectively alter structure and subsequently analyze function. We have adapted a method of overlap extension polymerase chain reaction (PCR) to generate multiple domain replacements in G-protein coupled receptors. The examples described herein are beta 2-adrenergic receptors whose G-protein coupling domains have been replaced by homologous domains of olfactory receptors, but the procedure has also been used to produce constructs with mutations, deletions, and fusions of two complete open reading frames. The chimeric olfactory-adrenergic receptors were assayed by functional expression in clonal lines of Xenopus melanophores. The ability of G-protein coupled second messenger pathways to cause translocation of pigment organelles within melanophores allows the use of video microscopy to assay the function of the chimeric receptors. Digital automation of microscope stage, camera, and image processing allows multiple parallel experiments to be performed. Melanophores allow responses mediated by the Gs, Gq and Gi pathways to be assayed with equal efficiency and the specificity of the coupling between chimera (or receptor) and G-protein subtypes can be rapidly determined.

Animals↗
PubMed2004

Human leucocyte antigen polymorphisms in nut-allergic patients in South Wales.

BACKGROUND: Peanuts and tree nuts are among the most common foods provoking severe allergic reactions including fatal anaphylaxis. However, little is known of the underlying genetic and immunological mechanisms involved. OBJECTIVE: Based on findings in other allergic diseases, we have investigated whether specific human leucocyte antigens (HLA) are associated with nut allergy. METHOD: Eighty-four patients presenting at the allergy clinic with symptoms of nut allergy were typed for the HLA Class I (HLA-A and B) and Class II (HLA-DRB1 and DQB1) loci by PCR using sequence-specific primers. Carriage frequencies were compared with 82 atopic non-nut-allergic subjects and 1798 random blood donors. RESULTS: The frequency of HLA-B(*)07 (28.57%) and DRB1(*)11 (15.48%) was increased in the nut-allergic patients compared to the atopic controls (12.20% and 3.66%, respectively) but not when compared to the blood donors (28.86% and 10.12%). DRB1(*)13 and DQB1(*)06 were both increased in frequency in the nut allergy patients over both the atopic and blood donor controls. However, none of these increased frequencies were significant when corrected for the number of comparisons undertaken. CONCLUSION: At HLA '2-digit resolution' and with undifferentiated patients with nut allergy, there are no major disturbances in the frequency of HLA-A, B, DRB1 or DQB1 types. However, the difference in frequency of HLA-DRB1(*)11 between the nut allergy patients and the atopic controls merits further investigation as this may represent an important phenotypic relationship.

Adolescent↗
PubMed2002

Digital analysis of cDNA abundance; expression profiling by means of restriction fragment fingerprinting.

BACKGROUND: Gene expression profiling among different tissues is of paramount interest in various areas of biomedical research. We have developed a novel method (DADA, Digital Analysis of cDNA Abundance), that calculates the relative abundance of genes in cDNA libraries. RESULTS: DADA is based upon multiple restriction fragment length analysis of pools of clones from cDNA libraries and the identification of gene-specific restriction fingerprints in the resulting complex fragment mixtures. A specific cDNA cloning vector had to be constructed that governed missing or incomplete cDNA inserts which would generate misleading fingerprints in standard cloning vectors. Double stranded cDNA was synthesized using an anchored oligo dT primer, uni-directionally inserted into the DADA vector and cDNA libraries were constructed in E. coli. The cDNA fingerprints were generated in a PCR-free procedure that allows for parallel plasmid preparation, labeling, restriction digest and fragment separation of pools of 96 colonies each. This multiplexing significantly enhanced the throughput in comparison to sequence-based methods (e.g. EST approach). The data of the fragment mixtures were integrated into a relational database system and queried with fingerprints experimentally produced by analyzing single colonies. Due to limited predictability of the position of DNA fragments on the polyacrylamid gels of a given size, fingerprints derived solely from cDNA sequences were not accurate enough to be used for the analysis. We applied DADA to the analysis of gene expression profiles in a model for impaired wound healing (treatment of mice with dexamethasone). CONCLUSIONS: The method proved to be capable of identifying pharmacologically relevant target genes that had not been identified by other standard methods routinely used to find differentially expressed genes. Due to the above mentioned limited predictability of the fingerprints, the method was yet tested only with a limited number of experimentally determined fingerprints and was able to detect differences in gene expression of transcripts representing 0.05% of the total mRNA population (e.g. medium abundant gene transcripts).

Journal Article↗
PubMed2005

Cloning and characterization of full length of a novel zebrafish gene Zsrg abundantly expressed in the germline stem cells.

Using the digital differential display program of the National Center for Biotechnology Information, we identified a contig of expression sequence tags (ESTs) (Accession No. BM316936), which came from zebrafish ovary and testis libraries. The full-length cDNA of this transcript was cloned and further confirmed by polymerase chain reaction and sequencing. The full-length cDNA of the novel gene is 807bp and encodes a novel protein of 187 amino acids, which shares no significant homology with any other known proteins. Characterization of genomic sequences of the gene revealed that it spans 6kb on the linkage group 3 and is composed of five exons and four introns. RT-PCR analysis showed that it was expressed in mature oocytes and one-cell stage, and persisted until 24h of development. RT-PCR also revealed that it is expressed in gonad and kidney, with the highest level of expression in the testis. The expression sites of the novel gene in adult gonad were further localized by in situ hybridization to oogonia and growing oocytes in ovary and to spermatogonia, spermatocytes but not to spermatids in testis. Based on its abundance in testis and the germline stem cell-spermatogonia and oogonia, we hypothesize that it may function as a testicular development and gametogenesis related gene that plays important roles in spermatogenesis, and named it Zsrg (zebrafish testis spermatogenesis related gene, Zsrg).

Amino Acid Sequence↗
PubMed2006

Net1 and Myeov: computationally identified mediators of gastric cancer.

Gastric adenocarcinoma (GA) is a significant cause of mortality worldwide. The molecular mechanisms of GA remain poorly characterised. Our aim was to characterise the functional activity of the computationally identified genes, NET 1 and MYEOV in GA. Digital Differential Display was used to identify genes altered expression in GA-derived EST libraries. mRNA levels of a subset of genes were quantitated by qPCR in a panel of cell lines and tumour tissue. The effect of pro- and anti-inflammatory stimuli on gene expression was investigated. Cell proliferation and invasion were measured using in an in-vitro GA model following inhibition of expression using siRNA. In all, 23 genes not previously reported in association with GA were identified. Two genes, Net1 and Myeov, were selected for further analysis and increased expression was detected in GA tissue compared to paired normal tissue using quantitative PCR. siRNA-mediated downregulation of Net1 and Myeov resulted in decreased proliferation and invasion of gastric cancer cells in vitro. These functional studies highlight a putative role for NET1 and Myeov in the development and progression of gastric cancer. These genes may provide important targets for intervention in GA, evidenced by their role in promoting invasion and proliferation, key phenotypic hallmarks of cancer cells.

Adenocarcinoma↗
PubMed2005

The occurrence of treponemes in contagious ovine digital dermatitis and the characterisation of associated Dichelobacter nodosus.

Contagious ovine digital dermatitis (CODD) is a recently recorded, apparently new infection of the ovine hoof, which differs clinically from footrot caused by Dichelobacter nodosus and which fails to respond well to accepted treatment practices for footrot. Despite the welfare implications of such an infection, very little research has been performed on CODD to date and the aetiology remains confused. Suggestions have been made that there is a potential role for treponemes in the pathogenesis of CODD but that D. nodosus is apparently not involved. Six farms were therefore targeted in this study to provide a more in-depth investigation into the bacterial flora of CODD lesions. Dark ground microscopy, culture and PCR techniques were used, concentrating on the presence of D. nodosus and spirochaetes, particularly those of the genus Treponema. The results demonstrated that isolates of D. nodosus were indeed present in a high percentage (74%) of CODD lesions compared with 31% of apparently healthy feet. The isolates were shown to be of similar virulence type to those reported previously in cases of footrot, and the range of serogroups was also found to be similar to footrot, with serogroup H being prevalent. Treponemes were present in 70% of CODD lesions and 38% of apparently healthy feet, supporting a possible association between CODD and treponemes. However, any further progress on the aetiology of CODD and the potential for novel, effective treatment will depend on an improved ability to culture these organisms routinely in the laboratory thereby enabling their complete characterisation.

Animals↗