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PubMed1999

Effect of the Factor V Leiden mutation on the severity of meningococcal disease.

BACKGROUND: One of the most serious complications of meningococcal disease is the syndrome of purpura fulminans, which is characterized by intravascular thrombosis and hemorrhagic infarction of skin, limbs and digits. The reasons why some patients with meningococcal disease develop purpura fulminans while others have minimal thrombotic and skin involvement despite having profound septic shock are not yet understood. The Factor V Leiden mutation (FV(L)) is associated with thrombotic events, and we hypothesized that children carrying FV(L) who develop meningococcal disease may be at increased risk of purpura fulminans. METHODS: We determined the FV(L) genotype by PCR and restriction enzyme digestion (Mnl1) in 259 children with meningococcal disease and 80 healthy controls. In addition 79 parents of children with fatal meningococcal disease were studied. RESULTS: There was no significant increase in the frequency of FV(L) in patients with meningococcal disease (10%) as compared with healthy controls (9%) or with the parents of children who died of meningococcal disease (12%). Although the mortality was not increased in patients heterozygous for FV(L), they had increased complications of purpura fulminans, as assessed by requirement for skin grafting, referral to plastic surgeon and/or amputation. Among survivors 5 of 24 (21%) of those heterozygous for FV(L) had complications, compared with 14 of 233 (7%) who were wild type [P < 0.03; relative risk, 3.1 (95% confidence intervals, 1.2 to 7.9)]. CONCLUSIONS: FV(L) exacerbates purpura fulminans in meningococcal disease but does not have a significant effect on mortality.

Child↗
PubMed1999

Confirmation of linkage of type 1 hereditary sensory neuropathy to human chromosome 9q22.

OBJECTIVES: 1) To confirm linkage of hereditary sensory neuropathy type 1 (HSN-I) to human chromosome 9q22 in a large American family of German origin. 2) To construct a yeast artificial chromosome (YAC) contig spanning the HSN-I candidate interval. 3) To investigate the HSN-I contig for potential candidate genes. BACKGROUND: HSN-I is a rare peripheral neuropathy characterized by loss of temperature sensation, ulceration and osteomyelitis of the digits, and subtle distal weakness. A gene for HSN-I has previously been mapped to human chromosome 9q22.1-q22.3 between markers D9S318 and D9S176 in an 8-cM interval in four Australian families. METHODS: In a large German-American family with HSN-I, genome-wide linkage analysis was performed on 68 family members extending over five generations and including 17 affected members. Genotyping was performed with PCR, and the resulting genotypes were analyzed with two-point linkage analysis with Fastlink. A YAC contig was constructed based on the Whitehead Institute YAC contig WC9.3. RESULTS: Two-point linkage analysis resulted in a maximum lod score of 8.2 at theta = 0 for marker D9S1815. Haplotype analysis locates the HSN-I gene between markers D9S1797 and D9S197. Using YAC clones from the Centre d'Etude du Polymorphism Humain YAC Library, we constructed a YAC contig spanning these markers. Based on the radiation hybrid map of the human genome, we estimate that the size of this interval is less than 2,500 kb. CONCLUSIONS: Our study confirms linkage of a putative HSN-I gene to chromosome 9q22, considerably narrows the HSN-I locus, and provides a basis for identification of the HSN-I gene.

Chromosome Mapping↗
PubMed2003

Positive correlation between single or combined genotypes of CYP1A1 and GSTM1 in relation to prostate cancer in Chilean people.

BACKGROUND: The prostate cancer is a slowly progressing disease that begins decades prior to diagnosis. It has been suggested that there might be differences in susceptibility due to genetic polymorphisms in biotransformation enzyme genes. In the present work, associations between CYP1A1(Msp1), GSTM1(-/-) polymorphisms, and prostate cancer were analyzed in a case-control study. METHODS: Genomic DNA was isolated from peripheral blood samples, collected on EDTA. PCR-RFLP was used to determine simultaneously Msp1 and GSTM1(-/-) polymorphisms. RESULTS: In cancer patients, frequency of m2 variant allele (0.377) and GSTM1(-/-) (0.362) showed statistically significant increases compared to the control group (0.262 and 0.227, respectively). The estimate relative risks (OR) were higher for individuals carrying combined CYP1A1 and GSTM1 rare genotypes, in relation to individuals carrying CYP1A1 or GSTM1 alone. Multivariate logistic regression analysis including confounding factors (age, digital examination, and PSA antigen) showed even higher risk for individuals carrying m2m2 genotype (OR = 3.99; 95% CI, 1.27-12.54), GST(-/-) genotype (OR = 2.75; 95% CI, 1.31-5.79), and m2m2/GST(-) genotype (OR = 16.63; 95% CI, 1.67-165.48). CONCLUSIONS: Taken together, these findings suggest that Chilean people carrying single or combined GSTM1 and CYP1A1 polymorphisms are more susceptible to prostate cancer.

Aged↗
PubMedDate not supplied

Spectral karyotyping, a 24-colour FISH technique for the identification of chromosomal rearrangements.

Spectral karyotyping (SKY) is a new fluorescence in situ hybridisation (FISH) technique that refers to the molecular cytogenetic analysis of metaphase preparations by means of spectral microscopy. For SKY of human metaphase chromosomes, 24 chromosome-specific painting probes are used in just one FISH experiment. The probes are labelled by degenerate oligonucleotide-primed PCR using three fluorochromes and two haptens. Each probe is differentially labelled with one, two, three or four fluorescent dyes, resulting in a unique spectral signature for every chromosome. After in situ hybridisation and immunodetection, a spectral image is acquired using a conventional fluorescence light microscope equipped with a custom-designed triple-bandpass filter and the SpectraCube, which is able to retrieve spectral information for every pixel in a digital CCD image. The 24-colour display and chromosome classification are based on the unique emission spectra of the chromosomes. Together with chromosome banding information from an inverted DAPI or a G-banded metaphase, a comprehensive overview of chromosomal aberrations is presented.

Color↗
PubMed2000

Quantitative microscopy after fluorescence in situ hybridization - a comparison between repeat-depleted and non-depleted DNA probes.

Complex probes used in fluorescence in situ hybridization (FISH) usually contain repetitive DNA sequences. For chromosome painting, in situ suppression of these repetitive DNA sequences has been well established. Standard painting protocols require large amounts of an unlabeled 'blocking agent', for instance Cot-1 DNA. Recently, it has become possible to remove repetitive DNA sequences from library probes by means of magnetic purification and affinity PCR. Such a 'repeat depleted library probe' was hybridized to the q-arm of chromosome 15 of human metaphase spreads and interphase cell nuclei without any preannealing by Cot-1 DNA. Apart from this, 'standard' FISH conditions were used. After in situ hybridization, microscope images were obtained comparable to those achieved with the #15q library probe prior to depletion. The images were recorded by a true color CCD camera. By digital image analysis using 'line scan' and 'area scan' procedures, the painting efficiency expressed in terms of relative fluorescence signal intensity was quantitatively evaluated. The painting efficiency using the repeat depleted probe of chromosome 15q was compared to the painting efficiency after standard FISH. The results indicate that both types of probes are compatible to a high FISH efficiency. Using equivalent probe concentrations, no significant differences were found for FISH with standard painting probes and repeat depleted painting probes.

Cell Nucleus↗
PubMed2003

OFD1, the gene mutated in oral-facial-digital syndrome type 1, is expressed in the metanephros and in human embryonic renal mesenchymal cells.

Oral-facial-digital syndrome type 1 (OFD1) causes polycystic kidney disease (PKD) and malformations of the mouth, face and digits. Recently, a gene on Xp22, OFD1, was reported to be mutated in a limited set of OFD1 patients. This study describes mutation analysis in six further OFD1 families. Additionally, gene expression was sought in human development. In two OFD1 kindreds affected by PKD, a frameshift mutation and a splice-site mutation were detected. In four apparently sporadic cases, three frameshift and a missense mutation were found. Using RT-PCR of RNA from first-trimester normal human embryos, both alternative splice forms of mRNA (OFD1a and OFD1b) were found to be widely expressed in organogenesis. Northern blot detected OFD1 mRNA in metanephros, brain, tongue, and limb, all organs affected in the syndrome. A polyclonal antibody directed to a C-terminal OFD1a epitope detected a 120-kD protein in the metanephros and in human renal mesenchymal cell lines. In normal human embryos, OFD1a immunolocalized to the metanephric mesenchyme, oral mucosa, nasal and cranial cartilage, and brain. Moreover, using normal human renal mesenchymal cell lines, the immunoreactive protein colocalized with gamma-tubulin, suggesting that OFD1 is associated with the centrosome. First, it is concluded that OFD1 mutations would generally be predicted to result in unstable transcripts or nonfunctional proteins. Second, OFD1 is expressed in human organogenesis; on the basis of the metanephric expression pattern, the results suggest that OFD1 plays a role in differentiation of metanephric precursor cells.

Cell Differentiation↗
PubMed1994

Resonant activation of calcium signal transduction in neurons.

The relevant parameters of calcium fluxes mediating activation of immediate-early genes and the collapse of growth cones in mouse DRG neurons in response to action potentials delivered in different temporal patterns were measured in a multicompartment cell culture preparation using digital fluorescence videomicroscopy. Growth cone collapse was produced by trains of action potentials causing a large rise in [Ca2+]i, but after chronic exposure to patterned stimulation growth cones regenerated and became insensitive to the stimulus-induced increase in [Ca2+]i. Calcium reached similar peak concentrations, but the [Ca2+]i increased more slowly than in naive growth cones (time constant of 6.0 s versus 1.4 s in naive growth cones). Semiquantitative PCR measurements of gene expression showed that pulsed stimulation delivered at 1-min intervals for 30 min induced expression of c-fos, but the same total number of action potentials delivered at 2-min intervals failed to induce c-fos expression, even though this stimulus induces a larger peak [Ca2+]i than the effective stimulus pattern. The experiments suggest that the kinetics of calcium fluxes produced by different patterns of stimulation, and changes in the kinetics of calcium flux in neurons under different states of activation, are critical in determining the effects of action potentials on growth cone motility or expression of IE genes during development of neuronal circuits. We propose that differences in kinetics of individual reactions in the stimulus-response pathway may lead to resonance of activation in the neuron, such that certain processes will be selectively activated by particular temporal patterns of stimulation.

Action Potentials↗
PubMed1992

PCR amplification and simultaneous digoxigenin incorporation of long DNA probes for fluorescence in situ hybridization.

Nonradioactive in situ hybridization has found widespread applications in cytogenetics. Basic requirements are DNA probes in sufficient amounts and of high specificity as well as a labeling protocol of good reproducibility. The PCR has been of fundamental importance for the amplification of DNA sequences and thus for the production of DNA probes. Meanwhile, PCR protocols for amplification of DNA have reached a high degree of automation. So far, incorporation of labeled nucleotides into these DNA probes has normally been done by nick translation. Here we show that in using the PCR, amplification of a DNA probe larger than one kilobase accompanied by simultaneous incorporation of digoxigenin-11-dUTP can be performed for in situ hybridization experiments. As an example, the DNA probe pUC 1.77 specific for the subcentromeric region q12 of chromosome number 1 was used and hybridized against metaphase chromosomes from human lymphocytes. The labeled chromosome region was detected by anti-digoxigenin-fluorescein, Fab fragments. The experiments were evaluated by digital image analysis of microphotographs.

Chromosomes, Human, Pair 1↗
PubMed1998

Longitudinal changes in TCRB variable gene expression and markers of gingival inflammation in experimental gingivitis.

The aim of this study was to gain information of the cellular and molecular events which occur during the development of experimental gingivitis and to determine whether such changes occur in the presence or absence of alveolar bone resorption. Clinical, radiographic, biochemical and immunological variables were monitored in a 3-week, single-centre, experimental gingivitis study of 10 healthy volunteers. Following screening and professional prophylaxis to achieve visibly healthy gingival status, subjects abstained from all oral hygiene practises in one maxillary (test) quadrant for a period of 21 days. At days 0 and 21, in test and (contralateral) control quadrants, % bleeding on controlled pressure probing (% BOP) was calculated, and radiographic alveolar bone status was assessed using bilateral standardised vertical bite-wing radiographs and digital subtraction radiography (DSR) analysis. In test quadrants, gingival crevicular fluid (GCF) was sampled from 4 sites per subject with Periopaper strips, and prostaglandin E2 (PGE2) levels measured using an enzyme immunoassay (EIA) kit. At days 0, 7 and 21, one interdental papilla was surgically excised from the test quadrant, and the expression of T cell receptor B variable (TCRBV) genes was investigated using a reverse transcription-polymerase chain reaction (RT-PCR) procedure. At days 0, 7 and 21, peripheral blood lymphocytes (PBL) were isolated and additionally investigated for TCRBV gene expression. Following 21 days of plaque accumulation in test quadrants, a statistically significant increase in % BOP scores confirmed the presence of gingival inflammation (p<0.001). DSR analysis revealed that there were no significant alveolar bone changes in either the test or control quadrants between days 0 and 21 (p>0.05). EIA analysis of GCF samples identified a significant decrease in mean GCF PGE2 concentrations from day 0 to day 21 (p<0.05). RT-PCR analysis indicated that genes from all 3 TCRBV families studied (TCRBV-2, -6, -8) were expressed in the PBL samples at all time points and in healthy gingival tissues at day 0. A restriction in the expression pattern of TCRBV genes similar to those which have previously been reported in chronic periodontitis was noted at gingivitis sites. It is possible that such an event may identify susceptibility to periodontal disease independently of other positive predictive markers such as GCF-PGE2.

Adolescent↗
PubMed1998

AFLP typing of Staphylococcus epidermidis in multiple sequential blood cultures.

AFLP is a novel high-resolution PCR-based DNA fingerprinting method generating complex banding patterns that can be used for comparative analysis. In the present study, the applicability of AFLP in fingerprinting Staphylococcus epidermidis isolates was investigated. The criteria considered were stability of patterns, reproducibility, discriminatory capacity and consistency with epidemiological context. Repeated testing of strains and investigation of subcultures showed that AFLP patterns were reproducible and stable with an intrastrain similarity of S > or = 94% as determined by analysis of digitized patterns. Fifteen unrelated strains were heterogeneous, with a level ranging from 78-93%. The applicability of AFLP in epidemiological studies of S. epidermidis was tested on 11 sets of four blood isolates each, from 11 patients with suspected septicaemia. Nine sets had indistinguishable or highly similar AFLP patterns for each isolate per set, while two sets had heterogeneous patterns. These results show that AFLP has high discriminatory power for strain identification in S. epidermidis.

Bacteremia↗
PubMed2004

Complete germline deletion of the STK11 gene in a family with Peutz-Jeghers syndrome.

Peutz-Jeghers syndrome (PJS, MIM175200) is an autosomal-dominant inherited disorder characterised by multiple gastrointestinal hamartomatous polyps, melanin spots of the oral mucosa and digits, and an increased risk for various neoplasms. The PJS results from germline alterations of the STK11/LKB1 tumour suppressor gene, located on 19p13.3, and encoding a serine/threonine kinase. The detection of STK11 germline mutations, in only 50-70% of PJS families, has suggested a genetic heterogeneity of the disease. We report the case of a family with typical features of PJS, including gastrointestinal hamartomatous, breast cancers and melanin spots of the oral mucosa. Quantitative multiplex PCR of short fluorescent fragments (QMPSF) of the 19p13 region allowed us to identify an approximately 250 kb heterozygous deletion removing entirely the STK11 locus. This report, which constitutes the first description of a complete germline deletion of STK11, shows that the presence of such large genomic deletions should be considered in PJS families without detectable point mutations of STK11.

AMP-Activated Protein Kinase Kinases↗
PubMed2024

Single Nucleotide Polymorphisms in RUNX2 and BMP2 contributes to different vertical facial profile.

The vertical facial profile is a crucial factor for facial harmony with significant implications for both aesthetic satisfaction and orthodontic treatment planning. However, the role of single nucleotide polymorphisms (SNPs) in the development of vertical facial proportions is still poorly understood. This study aimed to investigate the potential impact of some SNPs in genes associated with craniofacial bone development on the establishment of different vertical facial profiles. Vertical facial profiles were assessed by two senior orthodontists through pre-treatment digital lateral cephalograms. The vertical facial profile type was determined by recommended measurement according to the American Board of Orthodontics. Healthy orthodontic patients were divided into the following groups: "Normodivergent" (control group), "Hyperdivergent" and "Hypodivergent". Patients with a history of orthodontic or facial surgical intervention were excluded. Genomic DNA extracted from saliva samples was used for the genotyping of 7 SNPs in RUNX2, BMP2, BMP4 and SMAD6 genes using real-time polymerase chain reactions (PCR). The genotype distribution between groups was evaluated by uni- and multivariate analysis adjusted by age (alpha = 5%). A total of 272 patients were included, 158 (58.1%) were "Normodivergent", 68 (25.0%) were "Hyperdivergent", and 46 (16.9%) were "Hypodivergent". The SNPs rs1200425 (RUNX2) and rs1005464 (BMP2) were associated with a hyperdivergent vertical profile in uni- and multivariate analysis (p-value < 0.05). Synergistic effect was observed when evaluating both SNPs rs1200425- rs1005464 simultaneously (Prevalence Ratio = 4.0; 95% Confidence Interval = 1.2-13.4; p-value = 0.022). In conclusion, this study supports a link between genetic factors and the establishment of vertical facial profiles. SNPs in RUNX2 and BMP2 genes were identified as potential contributors to hyperdivergent facial profiles.

Polymorphism, Single Nucleotide↗
PubMed2004

Over-expression of gastrin-releasing peptide in human esophageal squamous cell carcinomas.

Gastrin-releasing peptide (GRP) is known as an autocrine growth factor for a number of gastrointestinal cancers. There is, however, little information on the expression of GRP in the squamous epithelia and squamous cell carcinoma, particularly in the esophagus. With a differential display approach, up-regulated GRP was observed in human esophageal squamous cell carcinoma (ESCC) samples obtained from a high-risk area for esophageal cancer, Linzhou in northern China. Up-regulation of phosphoglycerate mutase and P311 HUM (3.1) and down-regulation of keratin 13, cystatin B, endoglin and annexin I were observed. Using a reverse transcription-polymerase chain reaction (RT-PCR) method, significant over-expression of GRP was observed in 10 out of 12 ESCC samples (83.3%) and all four ESCC cell lines. With in situ hybridization, GRP mRNA expression was detected in nine out of 21 (42.8%) samples with basal cell hyperplasia (BCH), five out of seven (71.4%) samples with dysplasia (DYS) and 17 out of 24 (70.9%) ESCC samples. In contrast, GRP was expressed only in three out of 16 (18.7%) normal epithelium. Digital image analysis revealed that the mean value of GRP expression index, determined by intensity and area ratio of staining, was 0.19 in normal epithelium, 1.23 in BCH, 2.94 in DYS and 2.38 in ESCC, showing a progressive increase. Studies on ESCC cell lines showed GRP increased cell growth in a dose-dependent pattern in GRP receptor-positive ESCC cells, but not in GRP receptor-negative ESCC cells. GRP (1 mM) also increased cyclooxygenase-2 protein expression by 3.4-fold. This is the first demonstration that GRP is over-expressed in ESCC, and its over-expression may play a role in ESCC development and growth.

Base Sequence↗
PubMed1996

Highly sensitive nonradioactive single-strand conformational polymorphism. Detection of Ki-ras mutations.

Mutation detection by single-strand conformational polymorphism (SSCP) analysis is more difficult when the variant is limited to a small proportion of target sequences in a sample. Use of SYBR-Green II, a sensitive, nonradioactive, minimally hazardous nucleic acid stain, permits detection of Ki-ras mutants present as less than 0.5% of the target sequences. The polymerase chain reaction (PCR) primers we have selected produce an amplicon that distinguishes all clinically observed variants in Ki-ras codons 12 and 13 from the wild type. We compared mutant discrimination and SYBR-Green II detection sensitivity in three formats: (a) standard MDE gel SSCP, (b) rapid minigel MDE using an internal gel temperature controller, and (c) rapid resolution in chilled 15% (37.5:1) acrylamide minigels. All these gels are easily evaluated by standard ultraviolet transillumination and digital image analysis. This ssDNA staining method is rapid, highly reproducible, and minimally hazardous, and minigels use 25% the reagents of most other systems. Our improvements are relevant for the detection of mutations in pathologic samples with minimal targets, such as fine-needle aspirates, and body fluids in which mutated alleles of a gene may be present at low levels but carry a high level of diagnostic or prognostic importance.

DNA↗
PubMed2004

Role of lysophospholipid growth factors in the modulation of aqueous humor outflow facility.

PURPOSE: To investigate the role of lysophospholipid growth factors in the regulation of aqueous humor outflow in the trabecular meshwork (TM). METHODS: The expression profile of the endothelial differentiation gene (Edg) family of G-protein coupled receptors was determined by RT-PCR of human TM (HTM) cell-derived total RNA and by PCR amplification of HTM cell-derived and tissue-derived cDNA libraries. The effects of lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) on actin cytoskeleton and focal adhesions and on myosin light-chain (MLC) phosphorylation in HTM cells were evaluated by immunofluorescence microscopy and Western blot analysis, respectively. Activation of Rho GTPase in HTM cells was quantified by "pull-down" assays. Mobilization of intracellular calcium in HTM cells was determined using spectrofluorometric digital-imaging microscopy. The effects of LPA and S1P on aqueous humor outflow facility were evaluated by perfusion of enucleated porcine eyes. RESULTS: Each of the receptor isoforms Edg1, -2, -3, and -4 was readily detectable in three of four HTM cell-derived libraries, whereas Edg2 was detectable in the HTM tissue library. LPA (20 microM) and S1P (1 microM) stimulated actin stress fiber and focal adhesion formation, increased MLC phosphorylation, and induced marked activation of Rho GTPase in HTM cells. Both LPA (20 microM) and S1P (10 microM) also stimulated increases in intracellular calcium concentration in HTM cells. LPA- and S1P-induced effects on MLC phosphorylation in HTM cells were markedly inhibited by pretreatment with the Rho kinase-specific inhibitor Y-27632 (5 microM). Perfusion of LPA (50 microM) and S1P (5 microM) in enucleated porcine eyes produced a significant decrease in aqueous humor outflow facility from baseline of 37% (n = 6) and 31% (n = 5), respectively. CONCLUSIONS: These studies demonstrate that LPA and S1P, the physiological agonists of Edg receptors, decrease outflow facility in perfused porcine eyes in association with increased MLC phosphorylation and Rho guanosine triphosphatase (GTPase) activation. These data provide evidence for a novel mechanism for negative regulation of outflow facility, which may contribute to overall physiological homeostasis of aqueous humor outflow facility.

Actins↗
PubMed2003

Steroid hormone signaling between Schwann cells and neurons regulates the rate of myelin synthesis.

Fluorescence digital imaging microscopy was used to develop a method that allows the continuous monitoring and quantitative measurement of a single myelin internode throughout its development. Using this technique, steroid hormones such as progesterone and dexamethasone were shown to reduce the time required for the initiation and to regulate the rate of myelin synthesis. Progesterone was capable of increasing the rate of myelin synthesis in Schwann cell/neuronal co-cultures in a dose-dependent manner. RT-PCR and in situ hydridization studies revealed that the mRNAs for P450scc and 3beta-hydroxysteroid dehydrogenase, the enzymes involved in progesterone biosynthesis, were induced at the onset of myelin synthesis. The progesterone receptor protein translocated into the nucleus of the neurons during myelin synthesis, suggesting that progesterone could also be affecting neuronal gene expression. Changes in gene expression caused by progesterone are being examined to identify additional factors that may control myelin formation.

Animals↗
PubMed2003

Loss of clusterin both in serum and tissue correlates with the tumorigenesis of esophageal squamous cell carcinoma via proteomics approaches.

AIM: To identify the differentially secreted proteins or polypeptides associated with tumorigenesis of esophageal squamous cell carcinoma (ESCC) from serum and to find potential tumor secreted biomarkers. METHODS: Proteins from human ESCC tissue and its matched adjacent normal tissue; pre-surgery and post-surgery serum; and pre-surgery and normal control serum were separated by two-dimensional electrophoresis (2-DE) to identify differentially expressed proteins. The silver-stained 2-DE were scanned with digital ImageScanner and analyzed with ImageMaster 2D Elite 3.10 software. A cluster of protein spots differentially expressed were selected and identified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). One of the differentially expressed proteins, clusterin, was down-regulated in cancer tissue and pre-surgery serum, but it was reversed in post-surgery serum. The results were confirmed by semi-quantitative reverse-transcription (RT)-PCR and western blot. RESULTS: Comparisons of the protein spots identified on the 2-DE maps from human matched sera showed that some proteins were differentially expressed, with most of them showing no differences in composition, shape or density. Being analyzed by MALDI-TOF-MS and database searching, clusterin was differentially expressed and down-regulated in both cancer tissue and pre-surgery serum compared with their counterparts. The results were also validated by RT-PCR and western blot. CONCLUSION: The differentially expressed clusterin may play a key role during tumorigenesis of ESCC. The 2DE-MS based proteomic approach is one of the powerful tools for discovery of secreted markers from peripheral.

Aged↗
PubMed2002

Ligation-mediated PCR: robotic liquid handling for DNA damage and repair.

The investigation of in vivo DNA repair in mammalian cells at nucleotide resolution requires the quantification of break frequencies less than one per kilobase. By optimizing several parameters of the ligation-mediated PCR technique, we find that the required sensitivity can be achieved. We also report details of a one-day procedure that can be performed either with or without a robotic liquid handling workstation. The use of near-infrared fluorescent-labeled primers with detection by a LI-COR DNA sequencer provides for safe, nonradioactive detection, similar in sensitivity to the use of 32P-labeled primers but with the additional advantage that high-quality digitized data are obtained directly. Multiplexing can be performed; that is, more than one sequence can be analyzed in a single reaction, and multiple reactions can be processed robotically. Primer sets for exons 5-8 of the tumor suppressor gene, p53, were designed for simultaneous thermal cycling. The improved procedure with infrared detection was used to monitor low-frequency damage (<1 break/kb) and/or repair of UVB, UVC, and chemical methylation. Quantitative data on the linearity of response and reproducibility are described. The coefficient of variation for technical replicates was typically 10%. The methods described here will permit high sample throughput for the detection of DNA damage and repair as well as in vivo protein footprints.

Cells, Cultured↗