PubMed Health⌕ Search

Biomedical subjects

R Todd

Publications and source records attributed to R Todd.

At least 19 recordsLinked to original sources
PubMed2001

Oral cancer in vivo gene expression profiling assisted by laser capture microdissection and microarray analysis.

Large scale gene expression profiling was carried out on laser capture microdissected (LCM) tumor and normal oral epithelial cells and analysed on high-density oligonucleotide microarrays. About 600 genes were found to be oral cancer associated. These oral cancer associated genes include oncogenes, tumor suppressors, transcription factors, xenobiotic enzymes, metastatic proteins, differentiation markers, and genes that have not been implicated in oral cancer. The database created provides a verifiable global profile of gene expression during oral carcinogenesis, revealing the potential role of known genes as well as genes that have not been previously implicated in oral cancer.

Aged↗
PubMed2001

Induction of epithelial differentiation and DNA demethylation in hamster malignant oral keratinocyte by ornithine decarboxylase antizyme.

The hamster ornithine decarboxylase antizyme (ODC-Az) cDNA was transfected into the hamster malignant oral keratinocyte cell line, HCPC-1. Ectopic expression of ODC-Az resulted in the reversion of malignant phenotypes and alteration of DNA methylation status of CCGG sites. The phenotypes examined include ODC enzymatic activity, doubling time, morphological change, anchorage dependent growth, tumorigenicity in nude mice, induction of epithelial differentiation marker protein (involucrin), and change of cell cycle position. Comparison of CCGG DNA methylation status of the ODC-Az and control vector transfectants revealed a significant increase in demethylation of 5-methyl cytosines (m5C) of CCGG sites in the ODC-Az transfectants. Ectopic expression of ODC-Az gene in hamster malignant oral keratinocytes led to reduce ODC activity and the subsequent demethylation of 5-methyl cytosines, presumably via the ODC/ polyamines/ decarboxylated S-adenosylmethionine (dc-AdoMet) pathways. Our data suggest that ODC-Az shared the same pathway of polyamines/ dc-AdoMet/DNA methyltransferase (DNA MTase). We propose that ODC-Az mediates a novel mechanism in tumor suppression by DNA demethylation and presumably re-activation of key cellular genes silenced by DNA hypermethylation during cancer development. Oncogene (2001) 20, 24 - 33.

Agar↗
PubMed2001

Molecular characterization of a myelodysplasia-associated chromosome 7 inversion.

Chromosome 7 abnormalities are observed in a wide range of myeloid disorders, particularly myelodysplasia (MDS) and acute myeloid leukaemia (AML). Monosomy 7 and 7q deletions are the most frequent abnormalities, although translocations and inversions involving 7q also occur. The region 7q22--q34 may contain as many as four distinct minimal regions of deletion (MDRs), which are thought to contain one or more myeloid tumour-suppressor genes. We have defined previously the proximal breakpoint of a constitutional 7q22--q34 inversion, carried in a cell line derived from a member of a family with a history of MDS. A YAC clone spanning this breakpoint was identified. Both inversion breakpoints have now been cloned and sequenced, placing the proximal breakpoint 40 kb centromeric to the TAC2 (tachykinin 2) gene and the distal breakpoint 42 kb telomeric to the SSBP (mitochondrial single-stranded DNA-binding protein) gene. Sequence alignments revealed small (3--4 bp) duplications at the inversion breakpoints, suggesting that the mechanism of inversion involved the creation of staggered breaks and filling in of the overhanging ends. A 190-bp Alu--Alu deletion close to the distal breakpoint was also detected and may have contributed to the inversion.

Autoradiography↗
PubMed2001

From the chromosome to DNA: Restriction fragment length polymorphism analysis and its clinical application.

Understanding how chromosomal alterations contribute to acquired and inherited human disease requires the ability to manage the enormous physical and informational complexity of the deoxyribonucleic acid (DNA) packaged within. Important concepts and techniques involved in the analysis of DNA include restriction enzymes, Southern blotting, and restriction fragment length polymorphism/linkage analysis. These techniques have been essential in the understanding and diagnosis of several syndromes associated with the head and neck. The purpose of this article is to introduce DNA structure, describe some techniques fundamental to DNA analysis, and provide a brief overview of the clinical applications of this technology with respect to dentinogenesis imperfecta and oral field cancerization.

Blotting, Southern↗
PubMed2001

Reduction of p12DOC-1 expression is a negative prognostic indicator in patients with surgically resected oral squamous cell carcinoma.

PURPOSE: p12DOC-1 is a growth suppressor that negatively regulates cyclin-dependent kinase 2 (CDK2) activities. Expression of p12DOC-1 is reduced and/or lost in tumor tissues. The purpose of this study is to correlate in vivo the expression of p12DOC-1 in oral cancer tissues by immunohistochemistry with clinical and pathological parameters. EXPERIMENTAL DESIGN: Twenty-five cases of normal oral mucosa and 127 cases of oral squamous cell carcinomas were evaluated. Patients' charts were reviewed for clinical, pathological, and 10-year survival data. Because p12DOC-1 is a growth suppressor and associates with CDK2, parallel immunostaining was done for proliferating cell nuclear antigen and CDK2 to evaluate cell proliferation and potential correlation with CDK2. RESULTS: Our results showed that strong p12DOC-1 staining was uniformly seen in normal oral mucosa. p12DOC-1 staining was reduced or absent in 81 cases (63.8%) of oral squamous cell carcinomas. Decreased p12DOC-1 staining (<25% of cells stained) correlated with tumor mode of invasion (P = 0.001) and higher proliferating cell nuclear antigen (P = 0.0028) and CDK2 (P = 0.0020) expression. Survival analysis showed significant correlation of low p12DOC-1 expression with the risk of cervical lymph node metastasis (P = 0.001) and patients' 10-year survival status (P = 0.0214). CONCLUSIONS: These results allow us to conclude that reduction of p12DOC-1 protein expression is a frequent event in oral cancers. Intratumor immunohistochemical evaluation of p12DOC-1 expression can be an adjunctive prognostic indicator for patients with oral cancer.

Adult↗
PubMed2000

Expression of a truncated epidermal growth factor receptor in oral squamous cell carcinomas.

Epidermal growth factor receptor (EGFR) frequently overexpresses in cancers, including oral squamous cell carcinomas (OSCC). We previously identified a truncated EGFR (tEGFR) in human oral keratinocytes. In this study, we evaluated the prognostic value of tEGFR in 45 cases of OSCC. tEGFR expression inversely correlated with EGFR expression (r=-0.83, P<0.01), decreased with T-stage progression and lymph-node metastasis (P<0.05). The EGFR/tEGFR ratio correlated with the lymph-node metastasis (P<0.05) and survival outcome (hazard ratio =3.601; P<0.05). These results suggest that tEGFR may play an important roles in oral carcinogenesis and that the EGFR/tEGFR ratio may be a prognostic factor for OSCC.

Adult↗
PubMed2000

doc-1--mediated apoptosis in malignant hamster oral keratinocytes.

PURPOSE: Cell cycle mediators involved in inducing apoptosis are frequently deregulated during carcinogenesis. Deleted in oral cancer-1 (doc-1) is an S-phase regulator that is inactivated during oral carcinogenesis. Transfection of doc-1 into malignant oral keratinocytes leads to increased cell loss. It is hypothesized that ectopic expression of doc-1 in hamster oral cancer cells induces apoptosis. MATERIALS AND METHODS: Malignant hamster oral keratinocytes (wt-HCPC-1), which lack measurable doc-1 mRNA and protein, were previously transfected with either a CMV-doc-1 expression vector construct (doc-HCPC-1) or the parental control vector pcDNA3 (cv-HCPC-1). A trypan blue exclusion assay was performed to examine cell death in the parental or wild-type HCPC-1 keratinocytes, HCPC-1 transfected with the parental pcDNA3 vector, and the doc-1 transfected HCPC-1 cells. To examine whether ectopic expression of doc-1 mediates gross cellular changes consistent with apoptosis, toluidine blue-safranin differential staining and the quantitative fluorescent microscopy assays were performed. To identify early apoptotic cytochemical changes observed in the cell membrane and nucleus, annexin V/propidium iodide (PI) fluorescence-activated cell sorter (FACS) analysis and the terminal deoxytransferase-mediated dUTP nick-end labeling (TUNEL) assay were performed. RESULTS: Doc-HCPC-1 showed elevated numbers of dead cells over wt-HCPC-1 and cv-HCPC-1 in the trypan blue exclusion assay. Toluidine blue-safranin staining and quantitative fluorescent microscopy showed significant morphologic changes in the doc-1 transfectants consistent with apoptosis (P < .05). TUNEL assays (P < .05) and annexin V/PI FACS analysis (P < .05) also showed early cytochemical changes in the doc-HCPC-1 transfectants, confirming that ectopic expression of doc-1 induces apoptosis. CONCLUSIONS: These data suggest that doc-1 induces apoptosis in malignant hamster oral keratinocytes. It is hypothesized that doc-1 is a mediator of apoptosis that is inactivated during hamster oral carcinogenesis.

Animals↗
PubMed2000

Correction of the influence of baseline artefacts and electrode polarisation on dielectric spectra.

The deconvolution of biological dielectric spectra can be difficult enough with artefact-free spectra but is more problematic when machine baseline artefacts and electrode polarisation are present as well. In addition, these two sources of anomalies can be responsible for significant interference with dielectric biomass measurements made using one- or two-spot frequencies. The aim of this paper is to develop mathematical models of baseline artefacts and electrode polarisation which can be used to remove these anomalies from dielectric spectra in a way that can be easily implemented on-line and in real-time on the Biomass Monitor (BM). We show that both artefacts can be successfully removed in solutions of organic and inorganic ions; in animal cell and microbial culture media; and in yeast suspensions of varying biomass. The high quality of the compensations achieved were independent of whether gold and platinum electrodes were used; the electrode geometry; electrode fouling; current density; the type of BM; and of whether electrolytic cleaning pulses had been applied. In addition, the calibration experiments required could be done off-line using a simple aqueous KCl dilution series with the calibration constants being automatically calculated by a computer without the need for user intervention. The calibration values remained valid for a minimum of 3 months for the baseline model and indefinitely for the electrode polarisation one. Importantly, application of baseline correction prior to polarisation correction allowed the latter's application to the whole conductance range of the BM. These techniques are therefore exceptionally convenient to use under practical conditions.

Artifacts↗
PubMed2000

The chromosome: cytogenetic analysis and its clinical application.

PURPOSE: Chromosomes are highly structured, dynamic complexes of DNA and protein. The human genome consists of 46 chromosomes (22 paired autosomes and 2 sex chromosomes). Cytogenetic analysis provides a means to identify, as well as describe, chromosomes and disorder-related aberrations. The purpose of this article is to 1) introduce chromosome structure and alterations, 2) describe the technical fundamentals of cytogenetic analysis, and 3) provide a brief overview of the clinical applications of this technology.

Chromosomes, Human↗
PubMed2000

p12(DOC-1), a growth suppressor, associates with DNA polymerase alpha/primase.

p12(DOC-1) is a growth suppressor identified and isolated from normal keratinocytes. Ectopic expression of p12(DOC-1) in squamous carcinoma cells led to the reversion of in vitro transformation phenotypes including anchorage independence, doubling time, and morphology. Here we report that p12(DOC-1) associates with DNA polymerase alpha/primase (pol-alpha:primase) in vitro and in cells. The pol-alpha:primase binding domain in p12(DOC-1) is mapped to the amino-terminal six amino acid (MSYKPN). The biological effect of p12(DOC-1) on pol-alpha:primase was examined using in vitro DNA replication assays. Using the SV40 DNA replication assay, p12(DOC-1) suppresses DNA replication, leveling at approximately 50%. Similar results were obtained using the M13 single-stranded DNA synthesis assay. Analysis of the DNA replication products revealed that p12(DOC-1) affects the initiation step, not the elongation phase. The p12(DOC-1) suppression of DNA replication is likely to be mediated either by a direct inhibitory effect on pol-alpha:primase or by its effect on cyclin-dependent kinase 2 (CDK2), a recently identified p12(DOC-1)-associated protein known to stimulate DNA replication by phosphorylating pol-alpha:primase. p12(DOC-1) suppresses CDK2-mediated phosphorylation of pol-alpha:primase. These data support a role of p12(DOC-1) as a regulator of DNA replication by direct inhibition of pol-alpha:primase or by negatively regulating the CDK2-mediated phosphorylation of pol-alpha:primase.

Amino Acid Sequence↗
PubMed2000

p12(DOC-1) is a novel cyclin-dependent kinase 2-associated protein.

Regulated cyclin-dependent kinase (CDK) levels and activities are critical for the proper progression of the cell division cycle. p12(DOC-1) is a growth suppressor isolated from normal keratinocytes. We report that p12(DOC-1) associates with CDK2. More specifically, p12(DOC-1) associates with the monomeric nonphosphorylated form of CDK2 (p33CDK2). Ectopic expression of p12(DOC-1) resulted in decreased cellular CDK2 and reduced CDK2-associated kinase activities and was accompanied by a shift in the cell cycle positions of p12(DOC-1) transfectants ( upward arrow G(1) and downward arrow S). The p12(DOC-1)-mediated decrease of CDK2 was prevented if the p12(DOC-1) transfectants were grown in the presence of the proteosome inhibitor clasto-lactacystin beta-lactone, suggesting that p12(DOC-1) may target CDK2 for proteolysis. A CDK2 binding mutant was created and was found to revert p12(DOC-1)-mediated, CDK2-associated cell cycle phenotypes. These data support p12(DOC-1) as a specific CDK2-associated protein that negatively regulates CDK2 activities by sequestering the monomeric pool of CDK2 and/or targets CDK2 for proteolysis, reducing the active pool of CDK2.

Blotting, Western↗
PubMed2000

Laser capture microdissection-generated target sample for high-density oligonucleotide array hybridization.

Current advances in biomolecular technology allow precise genetic fingerprinting of specific cells responsible for the pathogenesis of human diseases. This study demonstrates the feasibility of generating target samples from laser capture microdissection (LCM) tissues suitable for hybridization of high-density oligonucleotide arrays for gene expression profiling. RNA was successfully isolated by LCM from three paired specimens of oral cancer and linearly amplified using T7 RNA polymerase. Evaluation of the cDNA revealed that five of five cellular maintenance transcripts are detected. Biotinylated cRNA was generated and hybridized to the human Test 1 GeneChip probe arrays, which demonstrated that the RNA is of sufficient quality and integrity to warrant further analysis. Subsequent hybridization of the samples to the HuGenFL GeneChip probe arrays revealed that 26.5%-33.0% of the approximately 7000 represented genes are expressed in each of the six samples. These results demonstrate that LCM-generated tissues can generate sufficient quality cRNA for high-density oligonucleotide microarray analysis, an important step in determining comprehensive gene expression profiling using this high-throughput technology.

Biotinylation↗
PubMed1999

Intragenic mutation analysis of the human epidermal growth factor receptor (EGFR) gene in malignant human oral keratinocytes.

Alteration in epidermal growth factor receptor (EGFR) expression is frequently associated with malignant transformation of epithelial tissues, including oral mucosa. This study examines the mutations in the coding region of the human EGFR gene in normal and malignant human oral keratinocytes. To examine the intragenic mutations in the human EGFR gene, a panel of normal and malignant human oral keratinocytes were examined by a nonisotopic RNase cleavage assay. Two consistent alterations were detected. First, a polymorphism, which generates a unique BsrI restriction site, was detected at position 2073. This BsrI polymorphism was present only in malignant keratinocytes. Second, Southern blot hybridization of PCR products revealed that there is a truncated EGFR mRNA (approximately 1.5-kb) in oral squamous cell carcinoma cell lines. Similar analysis in normal cell lines revealed that this truncated EGFR transcript is also present. Immunoblotting revealed the presence of this truncated form of EGFR in all keratinocyte cell lines. These data permit us to conclude that there exists a novel truncated form of EGFR in human oral keratinocytes. Furthermore, there exists a tumor-associated BsrI polymorphic site at position 2073. The potential biological relevance of the truncated receptor and the utility of the BsrI polymorphic site for diagnostic applications are currently being explored.

Carcinoma, Squamous Cell↗
PubMed1999

From concept to market in industrial impedance applications.

This paper discusses some of the technical aspects of converting a laboratory idea into a commercial product. The example used is the development of the Aber Instruments Biomass Monitor, which is now used worldwide in industry for the pitching of yeast in breweries and for biomass measurements in the pharmaceutical industry. Although the issues raised will relate to instrumentation in a production environment, many of the themes will be equally applicable to medical instruments.

Biomass↗
PubMed1999

Identification and mutation analysis of DOC-1R, a DOC-1 growth suppressor-related gene.

The tumor suppressor gene MEN1 and several oncogenes including CCND1/cyclin D1/PRAD1 map to chromosome 11q13. However, molecular and cytogenetic analysis suggests the presence of a second tumor suppressor locus at this chromosome region. We have identified a novel gene from chromosome 11q13, which encodes a protein of 126 amino acids sharing an overall 57% identity with the p12(DOC-1) protein encoded by the DOC-1 gene, the human homolog of hamster putative tumor suppressor doc-1 (deleted in oral cancer-1). We therefore designated the novel gene as DOC-1R for DOC-1-related. The cytogenetic location was confirmed by chromosome fluorescent in situ hybridization. Northern blot analysis indicated that it was expressed in all the tissues examined. DOC-1R protein showed heterogeneous subcellular localization. RT-PCR-SSCP analysis failed to detect deleterious mutations of the DOC-1R transcript in four premalignant oral keratinocyte lines and 20 different cancer cell lines from tumor types which frequently harbor LOH at chromosome 11q13.

Amino Acid Sequence↗
PubMed1999

Familial encephalopathy with neuroserpin inclusion bodies.

We report on a new familial neurodegenerative disease with associated dementia that has presented clinically in the fifth decade, in both genders, and in each of several generations of a large family from New York State-a pattern of inheritance consistent with an autosomal dominant mode of transmission. A key pathological finding is the presence of neuronal inclusion bodies distributed throughout the gray matter of the cerebral cortex and in certain subcortical nuclei. These inclusions are distinct from any described previously and henceforth are identified as Collins bodies. The Collins bodies can be isolated by simple biochemical procedures and have a surprisingly simple composition; neuroserpin (a serine protease inhibitor) is their predominant component. An affinity-purified antibody against neuroserpin specifically labels the Collins bodies, confirming their chemical composition. Therefore, we propose a new disease entity-familial encephalopathy with neuroserpin inclusion bodies (FENIB). The conclusion that FENIB is a previously unrecognized neurodegenerative disease is supported by finding Collins bodies in a small kindred from Oregon with familial dementia who are unrelated to the New York family. The autosomal dominant inheritance strongly suggests that FENIB is caused by mutations in the neuroserpin gene, resulting in intracellular accumulation of the mutant protein.

Amino Acid Sequence↗
PubMed1999

Eosinophil-derived transforming growth factors (TGF-alpha and TGF-beta 1) in human periradicular lesions.

Inflammatory mediators of periradicular lesions are poorly understood. Transforming growth factors-alpha and -beta 1 (TGF-alpha and TGF-beta 1) have been linked with the cellular processes for both soft and hard tissue wound healing. The purpose of this study is to demonstrate the cellular sources of TGF-alpha and TGF-beta 1 mRNA and protein in periapical lesions by in situ hybridization and immunohistochemistry. Nine periapical granulomas and nine periapical cysts were examined. TGF-alpha mRNA and protein were not detectable in the granulomas examined. However, eosinophils surrounding the periapical cysts demonstrated both TGF-alpha mRNA and protein. The vast majority of eosinophils present in the periapical granulomas and cysts also demonstrated TGF-beta 1 mRNA and protein. Other cells producing TGF-beta 1 were lymphocytes, fibroblasts, and monocytes. The presence of wound repair cytokines, such as TGF-alpha and TGF-beta 1, suggests a mechanism by which the host inflammatory response may participate in the repair and remodeling of periapical tissues.

Adult↗