Fluorescence-based Analysis Research Articles

The XRCC3 Thr241Met polymorphism and breast cancer risk: a case–control study in a Thai population

The X-ray repair cross-complementing group 3 gene (XRCC3) belongs to a family of genes responsible for repairing DNA double-strand breaks caused by normal metabolic processes and exposure to ionizing radiation. Polymorphisms in DNA repair genes may alter an individual's capacity to repair damaged DNA and may lead to genetic instability and contribute to malignant transformation. We examined the role of a polymorphism in the XRCC3 gene (rs861529; codon 241: threonine to methionine change) in determining breast cancer risk in Thai women. The study population consisted of 507 breast cancer cases and 425 healthy women. The polymorphism was analysed by fluorescence-based melting curve analysis. The XRCC3 241Met allele was found to be uncommon in the Thai population (frequency 0.07 among cases and 0.05 among controls). Odds ratios (OR) adjusted for age, body mass index, age at menarche, family history of breast cancer, menopausal status, reproduction parameters, use of contraceptives, tobacco smoking, involuntary tobacco smoking, alcohol drinking, and education were calculated for the entire population as well as for pre- and postmenopausal women. There was a significant association between 241Met carrier status and breast cancer risk (OR 1.58, 95% confidence interval (CI) 1.02–2.44). Among postmenopausal women, a slightly higher OR (1.82, 95% CI 0.95–3.51) was found than among premenopausal women (OR 1.48, 95% CI 0.82–2.69). Our findings suggest that the XRCC3 Thr241Met polymorphism is likely to play a modifying role in the individual susceptibility to breast cancer among Thai women as already shown for women of European ancestry.

CHKA and PCYT1Agene polymorphisms, choline intake and spina bifida risk in a California population

BackgroundNeural tube defects (NTDs) are among the most common of all human congenital defects. Over the last two decades, accumulating evidence has made it clear that periconceptional intake of folic acid can significantly reduce the risk of NTD affected pregnancies. This beneficial effect may be related to the ability of folates to donate methyl groups for critical physiological reactions. Choline is an essential nutrient and it is also a methyl donor critical for the maintenance of cell membrane integrity and methyl metabolism. Perturbations in choline metabolism in vitro have been shown to induce NTDs in mouse embryos.MethodsThis study investigated whether single nucleotide polymorphisms (SNPs) in human choline kinase A (CHKA) gene and CTP:phosphocholine cytidylytransferase (PCYT1A) gene were risk factors for spina bifida. Fluorescence-based allelic discrimination analysis was performed for the two CHKA intronic SNPs hCV1562388 (rs7928739) and hCV1562393, and PCYT1A SNP rs939883 and rs3772109. The study population consisted of 103 infants with spina bifida and 338 non-malformed control infants who were born in selected California counties in the period 1989–1991.ResultsThe CHKA SNP hCV1562388 genotypes with at least one C allele were associated with a reduced risk of spina bifida (odds ratio = 0.60, 95%CI = 0.38–0.94). The PCYT1A SNP rs939883 genotype AA was associated with a twofold increased risk of spina bifida (odds ratio = 1.89, 95% CI = 0.97–3.67). These gene-only effects were not substantially modified by analytic consideration to maternal periconceptional choline intake.ConclusionOur analyses showed genotype effects of CHKA and PCYT1A genes on spina bifida risk, but did not show evidence of gene-nutrient interactions. The underlying mechanisms are yet to be resolved.

SULT1A1 genotype and susceptibility to colorectal cancer

Several procarcinogens that are present in cooked red meat and tobacco smoke are substrates for sulfotransferase 1A1 (SULT1A1). The association between environmental exposures and colorectal cancer risk may be modified by individual differences in the metabolism. Thus, we investigated the effect of a common polymorphism in the SULT1A1 gene associated with decreased enzyme activity on the susceptibility to colorectal cancer in a population-based case-control study. Patients (505) and 604 age- and sex-matched controls provided detailed risk factor information and were genotyped for SULT1A1 G638A using a fluorescence-based melting curve analysis method. Multivariate logistic regression analysis was used to estimate colorectal cancer risk associated with environmental exposures by SULT1A1 genotype. SULT1A1 genotype was not an independent risk factor for colorectal cancer. Risk of colorectal cancer associated with frequent consumption of red meat was significantly elevated among carriers of the SULT1A1*2 allele but not increased among subjects with the SULT1A1*1/*1 genotype (odds ratio (OR) 2.1, 95% confidence interval (CI) 1.1-4.1 and OR 1.0, 95% CI 0.5-2.1, respectively). Colorectal cancer risk associated with 30+ pack-years of active smoking was higher among carriers of the SULT1A1*2 allele (OR 1.7, 95% CI 1.0-3.2) than among individuals with the SULT1A1*1/*1 genotype (OR 1.1, 95% CI 0.6-2.1). Our results do not support a main effect of SULT1A1 genotype with regard to colorectal cancer but suggest that individuals with the low activity SULT1A1*2 allele may be at higher risk following carcinogen exposure than those with the SULT1A1*1/*1 genotype.

Effect of NAT1 and NAT2 Genetic Polymorphisms on Colorectal Cancer Risk Associated with Exposure to Tobacco Smoke and Meat Consumption

N-Acetyltransferases 1 and 2 (NAT1 and NAT2), both being highly polymorphic, are involved in the metabolism of aromatic and heterocyclic aromatic amines present in cigarette smoke and red meat cooked by high-temperature cooking techniques. We investigated the effect of differences in acetylation capacity, determined by NAT1 and NAT2 genotypes, on colorectal cancer risk associated with exposure to tobacco smoke or red meat consumption. In this population-based case-control study in Germany, 505 patients with incident colorectal cancer and 604 age- and sex-matched control individuals with genotyping data and detailed risk factor information were included. Genotyping of NAT1 and NAT2 genetic polymorphisms was done using a fluorescence-based melting curve analysis method. The association between genotypes, environmental exposures, and colorectal cancer risk was estimated using multivariate logistic regression. Colorectal cancer risk associated with active smoking was elevated after accumulation of 30(+) pack-years of smoking [odds ratio (OR), 1.4; 95% confidence interval (95% CI), 0.9-2.2] but not significantly modified by either NAT1 or NAT2 genotype. Exposure to environmental tobacco smoke was associated with an increased risk for colorectal cancer only among NAT2 fast acetylators (OR, 2.6; 95% CI, 1.1-5.9 for exposure in childhood and adulthood). Frequent consumption of red meat significantly increased colorectal cancer risk for the group comprising all NAT2 fast acetylators or carriers of the NAT1*10 allele (OR, 2.6; 95% CI, 1.1-6.1) but not among those with "slow" NAT1 and NAT2 genotypes. Our findings indicate that NAT1 and NAT2 genotypes may contribute jointly to individual susceptibility and that heterocyclic aromatic amines may play an important role in colorectal cancer associated with red meat and possibly also exposure to environmental tobacco smoke.

A trichloroacetic acid-acetone method greatly reduces infrared autofluorescence of protein extracts from plant tissue

Immunoblotting is used to determine many important characteristics of proteins. After electrophoretic separation, proteins are transferred to a membrane and reacted with a specific antibody. The antibody-protein complex is then visualized by radiographic, chromogenic, chemiluminescent, or more recently described fluorescence detection methods. Fluorescence-based detection offers some advantages over other approaches, including increased sensitivity, improved quantifiable range, and the ability to detect multiple antigens on the same blot. However, this technique is unavailable for analysis of green plant tissues by standard extraction methods because of contaminating autofluorescent pigments. We have compared 3 methods for extracting protein from plant tissue for use with infrared fluorescence-based immunoblot analysis. We report a trichloroacetic acid-acetone method that effectively eliminates autofluorescence while retaining the immunogenicity of a target protein.

Administration Route- and Immune Cell Activation-Dependent Tumor Eradication by IL12 electrotransfer

Injection of DNA via electric pulses into targeted tissues is referred to as electrotransfer. Intratumoral electrotransfer of the IL12 gene is more effective than intramuscular electrotransfer of the same gene in the eradication of established tumors. To understand the underlying immunological mechanisms, T cell infiltration, CTL activity, inhibition of angiogenesis, and transgene expression were analyzed using immunohistochemistry, fluorescence-based CTL analysis, Northern blot, and ELISA. In addition, the therapeutic effects of IL12 gene therapy were determined in immunocompetent, immune-cell-depleted, and immunodeficient mice. We found that intratumoral, but not intramuscular, electrotransfer of the IL12 gene induces CD8+ T cell infiltration, CTL activity, and tumor eradication. Tumor eradication by intratumoral IL12 gene electrotransfer requires both NK and T cells. The absence of either cell type will abrogate the intratumoral IL12 gene therapy-induced tumor eradication. Such a requirement explains why tumors cannot be eradicated by intramuscular electrotransfer of the IL12 gene. Only NK-cell-dependent, and not T-cell-dependent, anti-tumor effects are induced by intramuscular administration. Together, these results suggest that NK cells play an important role in both administration routes, mediating tumor growth inhibition, but T cells are specifically activated by intratumoral IL12 gene electrotransfer and not by the intramuscular route.

Ratiometric Singlet Oxygen Nano‐optodes and Their Use for Monitoring Photodynamic Therapy Nanoplatforms

Ratiometric photonic explorers for bioanalysis with biologically localized embedding (PEBBLE) nanoprobes have been developed for singlet oxygen, using organically modified silicate (ORMOSIL) nanoparticles as the matrix. A crucial aspect of these ratiometric singlet-oxygen fluorescent probes is their minute size. The ORMOSIL nanoparticles are prepared via a sol-gel-based process and the average diameter of the resultant particles is about 160 nm. These sensors incorporate the singlet-oxygen-sensitive 9,10-dimethyl anthracene as an indicator dye and a singlet-oxygen-insensitive dye, octaethylporphine, as a reference dye for ratiometric fluorescence-based analysis. We have found experimentally that these nanoprobes have much better sensitivity than does the conventional singlet-oxygen-free dye probe, anthracene-9,10-dipropionic acid disodium salt. The much longer lifetime of singlet oxygen in the ORMOSIL matrix, compared to aqueous solutions, in addition to the relatively high singlet oxygen solubility because of the highly permeable structure and the hydrophobic nature of the outer shell of the ORMOSIL nanoparticles, results in an excellent overall response to singlet oxygen. These nanoprobes have been used to monitor the singlet oxygen produced by "dynamic nanoplatforms" that were developed for photodynamic therapy. The singlet oxygen nanoprobes could potentially be used to quantify the singlet oxygen produced by macrophages.

Development of a S. cerevisiae whole cell biocatalyst for in vitro sialylation of oligosaccharides

Absence of sialylation on recombinant glycoproteins compromises their efficacy as therapeutic agents, as it results in rapid clearance from the human bloodstream. To circumvent this, several strategies are followed, including the implementation of a post-secretion glycosylation step. In this paper we describe the engineering of yeast cells expressing active surface exposed Trypanosoma cruzi trans-sialidase (TS) fused to the yeast Aga2 protein, and the use of this yeast in the sialylation of synthetic oligosaccharides. In an attempt to improve overall protein accessibility on the yeast surface, we abolished hyperglycosylation on the yeast cell wall proteins. This was achieved by disrupting the OCH1 gene of the TS surface expressing strain, which resulted in increased enzymatic activity. Using a fluorescence-based activity assay and DSA-FACE structural analysis, we obtained almost complete conversion to a fully sialylated acceptor, whereas in the wild type situation this conversion was only partial. Increasing protein accessibility on the yeast surface by modifying the glycosylation content thus proved to be a valuable approach in increasing the cell wall associated activity of an immobilised enzyme, hence resulting in a more effective biocatalyst system.

Methods for isolating circulating epithelial cells and criteria for their classification as carcinoma cells

Novel assay methods developed for the isolation and characterization of circulating tumor cells (CTC) of epithelial origin offer the potential of markers for the non-invasive gathering of clinical information relevant to the diagnosis, evolution and treatment of carcinoma. Of the numerous techniques currently used to analyze CTC, slide-based assays are perhaps the most common. While traditional combined immunocytochemical/brightfield microscopy systems continue to be the most frequently employed, fluorescence-based analysis is gaining in importance. This is partly because fluorescence microscopy analysis of slide-mounted CTC can provide simultaneously cytogenetic as well as morphologic and multiple phenotypic information. In particular, fluorescence microscopy analysis of slide-mounted CTC can accurately determine genetic changes at the chromosomal level in patients with recurrent disease. More importantly, by identifying genetic aberrations in CTC, it becomes possible to choose those patients most likely to benefit from a given treatment. The potential of this technique has already been demonstrated by employing fluorescence in situ hybridization (FISH) methods to measure expression of the HER2/neu gene in tissue from patients with breast carcinoma for the specific purpose of identifying those patients most likely to respond to Trastuzumab targeted therapy. Here, we review the major methodologies used in the preparation and analysis of the slide-based assays.

Biallelic Deletions in INK4 in Cutaneous Melanoma Are Common and Associated with Decreased Survival

Both the retinoblastoma and p53 pathways are often genetically altered in human cancers and their complex regulation is in part mediated by the three gene products p16, p14(ARF), and p15 of the INK4 locus on chromosome 9p21. Partial or complete biallelic deletions of the INK4 locus have been recognized in a variety of malignant tumors, including malignant melanoma. We have in the present study measured the frequency of INK4 deletions in a large number of melanoma metastases and determined their association with clinicopathologic variables and survival data. Quantitative real-time PCR, as well as fluorescence-based fragment analysis, has been used to perform measurements of the relative allelic concentrations of the INK4 genes in 112 human melanoma tumor samples from 86 patients. Thirty-eight of 86 melanoma patients (44%) had metastases with biallelic losses in INK4. Ten of 20 patients with multiple metastases showed similar deletion patterns in all analyzed tumors. There was no significant association between any of the clinicopathologic variables and loss of INK4. However, loss of INK4 had an adverse effect on median survival from time of diagnosis. Patients with tumors with diploid INK4 had a median survival of 142 months, whereas those with monoallelic or biallelic loss in INK4 had a median survival of only 47 months (P = 0.006). Our results point to homozygous deletions in the INK4 region as being one of the most common genetic alterations in malignant cutaneous melanoma. INK4 deletions are associated with an adverse prognosis.

SULT1A1 genotype, active and passive smoking, and breast cancer risk by age 50 years in a German case–control study

IntroductionSulfotransferase 1A1 (encoded by SULT1A1) is involved in the metabolism of procarcinogens such as heterocyclic amines and polycyclic aromatic hydrocarbons, both of which are present in tobacco smoke. We recently reported a differential effect of N-acetyltransferase (NAT) 2 genotype on the association between active and passive smoking and breast cancer. Additional investigation of a common SULT1A1 genetic polymorphism associated with reduced enzyme activity and stability might therefore provide deeper insight into the modification of breast cancer susceptibility.MethodsWe conducted a population-based case–control study in Germany. A total of 419 patients who had developed breast cancer by age 50 years and 884 age-matched control individuals, for whom risk factor information and detailed smoking history were available, were included in the analysis. Genotyping was performed using a fluorescence-based melting curve analysis method. Multivariate logistic regression analysis was used to estimate breast cancer risk associated with the SULT1A1 Arg213His polymorphism alone and in combination with NAT2 genotype in relation to smoking.ResultsThe overall risk for breast cancer in women who were carriers of at least one SULT1A1*2 allele was not significantly different from that for women with the SULT1A1*1/*1 genotype (adjusted odds ratio 0.83, 95% confidence interval 0.66–1.06). Risk for breast cancer with respect to several smoking variables did not differ substantially between carriers of the *2 allele and noncarriers. However, among NAT2 fast acetylators, the odds ratio associated with passive smoking only (3.23, 95% confidence interval 1.05–9.92) was elevated in homozygous carriers of the SULT1A1*1 allele but not in carriers of the SULT1A1*2 allele (odds ratio 1.28, 95% confidence interval 0.50–3.31).ConclusionWe found no evidence that the SULT1A1 genotype in itself modifies breast cancer risk associated with smoking in women up to age 50 years. In combination with NAT2 fast acetylator status, however, the SULT1A1*1/*1 genotype might increase breast cancer risk in women exposed to tobacco smoke.

Adenoviral Overexpression of the Glutamylcysteine Ligase Catalytic Subunit Protects Pancreatic Islets against Oxidative Stress

The catalytic subunit of glutamylcysteine ligase (GCLC) primarily regulates de novo synthesis of glutathione (GSH) in mammalian cells and is central to the antioxidant capacity of the cell. However, GCLC expression in pancreatic islets has not been previously examined. We designed experiments to ascertain whether GCLC is normally expressed in islets and whether it is up-regulated by interleukin-1 beta (IL-1 beta). GCLC expression levels were intermediate compared with other metabolic tissues (kidney, liver, muscle, fat, and lung). IL-1 beta up-regulated GCLC expression (10 ng/ml IL-1 beta, 3.76 +/- 0.86; 100 ng/ml IL-1 beta, 4.22 +/- 0.68-fold control) via the p38 form of mitogen-activated protein kinase and NF kappa B and also increased reactive oxygen species levels (10 ng/ml IL-1 beta, 5.41 +/- 1.8-fold control). This was accompanied by an increase in intraislet GSH/GSSG ratio (control, 7.1 +/- 0.1; 10 ng/ml IL-1 beta, 8.0 +/- 0.5; 100 ng/ml IL-1 beta, 8.2 +/- 0.5-fold control; p < 0.05). To determine whether overexpression of GCLC increases the antioxidant capacity of the islet and prevents the adverse effects of IL-1 beta on glucose-induced insulin secretion, islets were infected with an adenovirus encoding GCLC. IL-1 beta significantly decreased glucose-stimulated insulin secretion (control, 123.8 +/- 17.7; IL-1 beta, 40.2 +/- 3.9 microunits/ml insulin/islet). GCLC overexpression increased intraislet GSH levels and partially prevented the decrease in glucose-stimulated insulin secretion caused by IL-1 beta. These data provide the first report of GCLC expression in the islet and demonstrate that adenoviral overexpression of GCLC increases intracellular GSH levels and protects the beta cell from the adverse effects of IL-1 beta.

A Rare Mutation in the Primer Binding Region of the Amelogenin Gene Can Interfere with Gender Identification

PCR amplification of part of the X-Y homologous amelogenin gene with a single primer pair has been used as a sex identification test because it generates different length products from the X and Y chromosomes. Using a commercially available kit that contains amelogenin primers, we report a single phenotypically normal Caucasian male out of 327 males tested to date that failed to show an X chromosome-specific PCR product. Using alternative amelogenin primers external to but encompassing the initial amplicon, an X chromosome-specific product was seen. Sequence analysis of this X-specific PCR product revealed a C to G mutation at the most 3' base of the initial reverse amelogenin PCR primer. An alternative reverse PCR primer with this most 3' base deleted showed X- and Y-specific products from the case study male. Rare mutations that result in a failure to amplify sex chromosome-specific products can result in incorrect gender identification.

Loss of Heterozygosity Analysis of Benign, Atypical, and Anaplastic Meningiomas

Up to 70% of typical meningiomas demonstrate allelic loss at chromosome 22q. Allelic loss at additional chromosomal loci is associated with atypia and anaplasia in meningiomas. The pattern of allelic loss or loss of heterozygosity (LOH) follows a nonrandom, multistep pattern. All surgical meningioma samples obtained from 1991 to 1992 at the University of Pittsburgh Medical Center were analyzed according to current World Health Organization criteria. Samples without constitutional deoxyribonucleic acid (DNA) were excluded from this analysis. Individual hematoxylin and eosin slides from 43 patients were microdissected, and the DNA was harvested and amplified in the presence of 24 pairs of polymerase chain reaction primers, representing 24 microsatellite loci. The polymerase chain reaction products were subjected to capillary gel electrophoresis and a fluorescence-based DNA analysis system. LOH was defined as ratios of allelic peak heights falling within a conservative threshold of less than 0.5 or more than 2.0. Fisher's exact test and receiver operator characteristic curves were used to test the relationship between benign versus atypical and malignant pathological features and LOH at specific loci or combinations of loci. On review by two independent pathologists, 34 benign meningiomas, 6 atypical meningiomas, and 3 anaplastic meningiomas were identified. The mean number of alleles with LOH was 1.5 +/- 1.2 for benign meningiomas, 6.7 +/- 2.7 for atypical meningiomas, and 8.3 +/- 2.3 for anaplastic meningiomas (P < 0.001). The most important individual loci to predict malignancy were D1S407 (P = 0.006), L-myc (P < 0.001), D10S520 (P = 0.003), D10S1173 (P = 0.042), D11S1920 (P < 0.001), D14S555 (P = 0.041), D17S1289 (P < 0.001), D22S417 (P = 0.001), D22S431 (P = 0.019), and D22S532 (P = 0.028). Combining the LOH data across loci, the area under the receiver operator characteristic curve was 0.993, corresponding to virtually perfect prediction of pathological characteristics. Microsatellite marker analysis of allelic loss is a useful method of predicting atypia and anaplasia in meningiomas. More regions of allelic loss are seen in anaplastic and atypical meningiomas as compared with benign meningiomas. This study confirms previously reported chromosomal regions of allelic loss in atypical and anaplastic meningiomas and suggests additional chromosomal regions that may represent heretofore uncharacterized deletions within meningiomas. This type of genetic fingerprint ultimately may serve both a diagnostic and therapeutic role.

Rapid identification of the origin of the matrix protein gene in vaccine candidate reassortants using real-time PCR and fluorescence-based melting curve analysis

A simple and rapid method using real-time PCR and melting temperature analysis is described for the detection of the origin of the matrix gene in reassortant influenza A viruses. This procedure relies on differences between the matrix gene PCR product melting temperature of recent human A(H1N1) and A(H3N2) strains and the laboratory donor strain PR-8 allowing the origin of the matrix gene to be identified. This novel method offers advantages over previous methods such as hybridisation and RFLP in facilitating rapid selection of candidate vaccine strains.

588. A Central Role of RNA in Developing Therapeutic Vaccines for Cancer and Infectious Diseases

Intratumoral (tumor local) and intramuscular (systemic) immuno-electrogenetherapies are important for treating solid tumor and residual or metastatic tumors, respectively. However, intratumoral injection of IL-12 gene via electroporation is more effective in the inhibition of tumor growth than intramuscular injection, but the underlying mechanism that causes this administration route-dependent inhibition of tumor growth is not fully understood. To understand the cellular mechanism by which tumor growth is significantly inhibited by intratumoral but not by intramuscular IL-12 electro-gene therapies, induction of T-cell infiltration, CTL activity, inhibition of angiogenesis and transgene expression were determined using northern blot, ELISA, immunohistochemistry, and fluorescence-based CTL analysis, and the therapeutic effect was determined in immunocompetent, immune cell-depleted, and immunodeficient mice. The results indicated that intratumoral electric pulses, when combined with the injection of plasmid DNA, is one of the primary factors that results in the different therapeutic efficacies between intratumoral and intramuscular delivery. Intratumoral injection of control plasmid DNA via electric pulses yields a small tumor volume that is only 37% and 58% of the tumor volumes from mice treated with intramuscular injection of plasmid DNA without and with IL-12 via electroporation, respectively. Inclusion of IL-12 in the intratumoral injection further inhibits the tumor growth and yields a tumor volume that is 6% and 9% of the tumor volume from mice treated with intramuscular injection of control plasmid DNA and IL-12 gene, respectively. CD8 T-cells are the primary effector cells that are associated with this administration route-dependent tumor inhibition because intratumoral, but not intramuscular injection via electric pulses induces CD8+ T-cell infiltration and CTL activity, and because both intratumoral and intramuscular IL-12 electro-gene therapies induce similar levels of antiangiogenesis-and NK cell-mediated antitumor effect. These findings indicate that induction of tumor-specific CD8+ T-cell response is a prerequisite for improving the therapeutic effect mediated by the systemic immuno-electrogenetherapy.

Telomerase activation and expression of its catalytic subunits in benign and malignant tumors of the parathyroid.

Telomerase activity (TA) has been extensively studied in tumors of many organs, but not the parathyroid gland. Therefore, we investigated TA in parathyroid tumors, and examined the mRNA expression of its catalytic subunits. We examined 17 single adenomas, one hyperplastic parathyroid gland, and one metastatic parathyroid cancer and quantified TA by fluorescence-based TRAP analysis. We also studied the expression of telomerase catalytic subunits in nine adenomas and one cancer by reverse transcription - polymerase chain reaction (RT-PCR) analysis. Telomerase activity was not detected in any of the adenomas or the hyperplastic gland; however, the metastatic cancer was highly positive for TA. Both human telomerase RNA ( hTR) and human telomerase-associated pretein (hTEP-1) mRNA were detected universally in every specimen tested. Conversely, human telomerase reverse transcriptase (hTERT) mRNA was not detected by the conventional electrophoresis-based technique. Faint expression of hTERT mRNA was detected by real-time RT-PCR only in the sample of parathyroid cancer. We hypothesize that TA plays a role in the malignant transformation of parathyroid disease and suggest that hTERT mRNA expression could be the key step for TA as in other malignancies. Telomerase activity and hTERT may be useful molecular targets of parathyroid cancer.

Fluorescent-based BAT-26 analysis for distinct screening of microsatellite instability in colorectal cancers

For easy screening of genetic instability in colorectal cancers, we tried BAT-26 and BAT-25 mononucleotide repeats using fluorescent analysis and evaluated their usefulness and problems compared with other markers: D5S346, D17S250, D2S123, and D2S391, D4S392 (located near BAT-26 and BAT-25 respectively). The high-frequency of MSI (MSI-H) tumours, defined as tumours having instability in more than two markers, were detected in 8/146 (5.5%). These MSI-H cases were younger ages at diagnosis, and showed significantly higher incidences of right side location, and poorly differentiated histology than other cases (p<0.05). Four cases (2.7%) showed a 1 bp size shift in BAT-26 and 2 of them showed loss of heterozygosity (LOH) at D2S391 near BAT-26 locus. Among 9 cases with a 1 bp size shift in BAT-25, 6 cases showed LOH at D4S392 near the BAT-25 locus (p=0.035). In all 4 cases, non-cancerous DNA had two analogous peaks of BAT-26, indicating the heterozygosity of BAT-26 in constitutional DNA. This phenomenon was also detected in the peaks of BAT-25 in some cases, in whose constitutional DNAs, 1 bp size shift was also detectable in three other markers. To elucidate the reasons for the alterations of the 1 bp size shift of peak of these markers, we examined by microsatellite analysis mixed samples of tumour DNA with complete loss of the one allele at the 1p loci and each constitutional DNA sample of neuroblastoma patients. One base shift of the peak signal of the microsatellite marker was clearly obtained in proportion to the ratio of cancerous DNA and constitutional DNA. Fluorescent-based analysis of BAT-26 or BAT-25 was easy and useful for detection of MSI-H in colorectal cancers without analyzing non-cancerous DNA. A 1 bp size shift in BAT-26 or BAT-25 was considered to be affected by LOH at these loci. Thus, it is important to distinguish MSI from LOH to evaluated MSI using these markers.

Illinois Bundleflower Genetic Diversity Determined by AFLP Analysis

Illinois bundleflower [Desmanthus illinoensis (Michx.) MacMillan] is an herbaceous perennial legume native to North America. Useful as a N2–fixing plant in warm‐season grass pastures, Illinois bundleflower is also a promising perennial grain crop. Knowledge of the distribution of genetic variation in Illinois bundleflower would increase efficiency of germplasm preservation and expedite plant breeding progress. The objective of this experiment was to determine the distribution of genetic variation within and between Illinois bundleflower accessions by amplified fragment length polymorphism (AFLP) markers. Semi‐automated fluorescence‐based AFLP analysis was performed on three individuals from each of 50 accessions. We identified 222 markers, 159 of which were polymorphic. Within‐accession diversity was low (HS = 0.013) compared with total gene diversity (HT = 0.086). Analysis of molecular variance (AMOVA) showed that 83% of the molecular variance was explained by two major clusters, supporting a previous phenotypic study that suggested the existence of two distinct races of Illinois bundleflower. The less commonly occurring of the two races contained greater genetic diversity and originated from a localized region in the south central USA. The collection of more accessions from throughout the USA and focused on the Southeast would likely increase the genetic variation available to plant breeders.

Cell characterization of mononuclear and giant cells constituting pigmented villonodular synovitis

The aim of this study was to determine the histologic and cellular characteristics of 2 cell types, mononuclear cells (Mos) and multinuclear giant cells (GCs), that predominantly constitute pigmented villonodular synovitis (PVS). Synovial tissues examined in this study were obtained from 10 patients with PVS. Five methods were used for cell analysis: (1) enzyme-histochemistry for tartrate-resistant acid phosphatase (TRAP); (2) immunohistochemistry using antibodies for CD68, macrophage colony–stimulating factor (M-CSF), MIB-1, p53, p21, p16, and cathepsin-L (cath L); (3) TdT-mediated deoxyuridine triphosphate-biotin terminal end labeling (TUNEL) as a measure of apoptosis; (4) fluorescence-based polymerase chain reaction single-strand conformation polymorphism analyses (FPCR-SSCP) to detect p53 gene mutations; and (5) in situ hybridization using gene-specific oligoprobes for matrix metalloproteinase (MMP)-2, MMP-9, receptor activator of nuclear factor κB ligand (RANKL), and calcitonin receptor (CTR). Both Mos and GCs were shown to express the macrophage/histiocyte marker CD68. In GCs, TRAP and CTR, both of which are known as characteristic phenotype markers of osteoclasts, were expressed. M-CSF and RANKL, which are together essential for osteoclast differentiation, were expressed in both Mos and GCs. Mos were shown to express MIB-1, but GCs were not. Although proliferation-suppressor proteins p53, p21, and p16 were expressed in both Mos and GCs, little apoptotic phenomenon of lining Mos was detected by TUNEL. In our study, p53 gene mutations for exons 5, 7, and 8 in PVS synovial tissues were not detected by FPCR-SSCP analysis. Furthermore, both types of cells demonstrated the proteolytic enzymes MMP-2 and MMP-9 mRNA, and cath L protein. These results suggest that PVS has a hyperplastic property consisting of the CD68-positive monocytic cell lineage with differentiation of osteoclastic giant cells from monocyte and probably controlled against proliferation by wild-type p53, p21, and p16. HUM PATHOL 34:65-73. Copyright 2003, Elsevier Science (USA). All rights reserved.