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Biomedical subjects

K Truong

Publications and source records attributed to K Truong.

13 recordsLinked to original sources
PubMed2001

The use of FRET imaging microscopy to detect protein-protein interactions and protein conformational changes in vivo.

Intermolecular and intramolecular FRET between two spectrally overlapping green fluorescent protein variants fused to two different host proteins or at two different sites within the same protein offers a unique opportunity to monitor real-time protein-protein interactions or protein conformational changes. By using fluorescence digital imaging microscopy, one can visualize the location of green fluorescent proteins within a living cell and follow the time course of the changes in FRET corresponding to cellular events at a millisecond time resolution. The observation of such dynamic molecular events in vivo provides vital insight into the action of biological molecules.

Calcium Signaling↗
PubMed2001

FRET-based in vivo Ca2+ imaging by a new calmodulin-GFP fusion molecule.

Intracellular Ca2+ acts as a second messenger that regulates numerous physiological cellular phenomena including development, differentiation and apoptosis. Cameleons, a class of fluorescent indicators for Ca2+ based on green fluorescent proteins (GFPs) and calmodulin (CaM), have proven to be a useful tool in measuring free Ca2+ concentrations in living cells. Traditional cameleons, however, have a small dynamic range of fluorescence resonance energy transfer (FRET), making subtle changes in Ca2+ concentrations difficult to detect and study in some cells and organelles. Using the NMR structure of CaM bound to the CaM binding peptide derived from CaM-dependent kinase kinase (CKKp), we have rationally designed a new cameleon that displays a two-fold increase in the FRET dynamic range within the physiologically significant range of cytoplasmic Ca2+ concentration of 0.05-1 microM.

Animals↗
PubMed2000

Calmodulin target database.

The intracellular calcium sensor protein calmodulin (CaM) interacts with a large number of proteins to regulate their biological functions in response to calcium stimulus. This molecular recognition process is diverse in its mechanism, but can be grouped into several classes based on structural and sequence information. We have developed a web-based database (http://calcium.uhnres.utoronto.ca/ctdb) for this family of proteins containing CaM binding sites or, as we propose to call it herein, CaM recruitment signaling (CRS) motifs. At present the CRS motif found in approximately 180 protein sequences in the databases can be divided into four subclasses, each subclass representing a distinct structural mode of molecular recognition involving CaM. The database can predict a putative CRS location within a given protein sequence, identify the subclass to which it may belong, and structural and biophysical parameters such as hydrophobicity, hydrophobic moment, and propensity for alpha-helix formation.

Amino Acid Sequence↗
PubMed2000

Cadherins in embryonic and neural morphogenesis.

Cadherins not only maintain the structural integrity of cells and tissues but also control a wide array of cellular behaviours. They are instrumental for cell and tissue polarization, and they regulate cell movements such as cell sorting, cell migration and cell rearrangements. Cadherins may also contribute to neurite outgrowth and pathfinding, and to synaptic specificity and modulation in the central nervous system.

Amino Acid Sequence↗
PubMed1999

Evidence for in vitro selection during cell culturing of breast cancer: detection by flow and image cytometry.

Detailed studies of chromosome rearrangements within solid tumors require karyotype analysis after cell culturing. However, different cell subpopulations with various growth capacities within one tumor may introduce biases in karyotype analysis, known as the in vitro selection. In our laboratory, 22% of karyotypes from breast cancers established after short-term culture were normal. Using interphase fluorescence in situ hybridization (FISH) for the determination of chromosome 1 arm imbalances and flow cytometry measurements of ploidy, we demonstrated that at least 2/3 of these tumors were mainly composed of aneuploid cell populations. Thus, the incidence of normal or balanced karyotypes among breast cancers is probably below 7%. This is the first direct proof for the existence of an in vitro selection within breast cancer cultures, suggesting cautious interpretation of cytogenetic data.

Adult↗
PubMed1999

Quantitative fluorescence in situ hybridization in lung cancer as a diagnostic marker.

The diagnosis of lung cancer is quite often hampered by the existence of various cell types within samples such as biopsies or pleural effusions. We have established a new marker for image cytometry of interphase tumor cells of the lung by using the most recurrent and early cytogenetic event in lung cancer, the loss of the short arm of chromosome 3. The method is based on the detection of the imbalance between the long and the short arms of chromosome 3 by performing two-color fluorescence in situ hybridization on both arms. Fourteen tumors were analyzed after short-term culture and compared with the corresponding cytogenetic data obtained from metaphase analysis. Results on interphase nuclei and control experiments on metaphases were the same, with imbalance ratios ranging from 1.0 to 2.0 (mean value 1.6, median 1.5). To assess the clinical significance of this approach, three pleural effusions were analyzed. Data showed that normal cells within the sample could have been distinguished from the tumor cells based on different imbalance values between the long and the short arms. Thus, our method allows refined detection of lung tumor cells within samples containing heterogeneous cell populations.

Biomarkers, Tumor↗
PubMed1998

Fluorescence-based analysis of DNA ploidy and cell proliferation within fine-needle samplings of breast tumors: a new approach using automated image cytometry.

BACKGROUND: Automated image cytometry can allow concurrent quantification of several parameters in each individual cell within a population, opening new possibilities for diagnosis and prognosis. In this study, the authors investigated the capacity of this method for performing a bivariate analysis of DNA ploidy and synthesis in fine-needle samplings obtained without aspiration from breast tumors. METHODS: Samplings from 25 unselected cases of ductal infiltrative breast adenocarcinoma and 2 cases of fibroadenoma were analyzed. For each case, 3-5 slides (containing approximately 1000 cells each) were quantified to assess experimental precision. Ploidy was determined by fluorescent staining of DNA using 4,6-diamidino-2-phenylindole (DAPI). Contaminating lymphocytes were taken as internal controls to calculate DNA indices. DNA synthesis was analyzed by immunofluorescent detection of 5-bromodeoxyuridine (BrdU) incorporation. Measurements were compared with flow cytometric data obtained from the same patients. RESULTS: Relative error in determination of DNA indices was generally below 5%. Determination of proliferation indices were more variable, with a mean relative error of 25%. Two different populations of BrdU positive cells were detected systematically, one in the diploid and another in the aneuploid fraction. For both cytometric methods, DNA indices were similar in all 27 cases, whereas BrdU labeling indices showed no significant correlation in 13 cases. The remaining cases were not comparable due to lack of flow cytometric data. Labeling indices obtained by image cytometry did not reveal any significant correlation with Scarff-Bloom-Richardson grading or clinical staging. CONCLUSIONS: Automated image cytometry allows concurrent measurement of ploidy and cell proliferation within individual breast carcinoma cells. Statistical reliability can be reached with a relative small number of cells (1000), which is crucial for samples in which the cell number is too low for flow cytometry analysis. Visual control for artifact elimination and better characterization of cell populations makes this a powerful tool for tumor cell investigation. Automated image cytometry allows the obtainment of valuable prognostic parameters of traditional flow cytometry with the relatively small number of cells obtained in aspiration procedures.

Adult↗
PubMed1998

Quantitative FISH by image cytometry for the detection of chromosome 1 imbalances in breast cancer: a novel approach analyzing chromosome rearrangements within interphase nuclei.

Interphase cytogenetics have become a widespread tool for investigation of chromosome rearrangements in solid tumors. The most recurrent chromosome alteration within breast cancer affects chromosome 1, leading principally to gain of the long arm and/or loss of the short arm. We have developed a new method for detection of chromosome 1 arm imbalances in interphase nuclei. The method is based on quantitation of the fluorescence signals emitted by the hybridized two-color paintings of the short and long arms using image cytometry. The chromosome arm imbalance was determined by calculating the ratio of both fluorescence emissions of each arm. The ratio of the paintings of normal lymphocytes was used as a reference. Three breast cancer cell lines, 13 fresh tumor samples, and 6 fine-needle samplings of breast cancer were analyzed using an automated image cytometer. Whenever possible, classic cytogenetics and in situ hybridization on metaphases were performed as controls. Fluorescence ratios representing the imbalances of chromosome 1 arms with values between 1 and 3.2 were measured. Data between classic cytogenetics and interphase cytogenetics were well-correlated (r = 0.89). This method, which enables an easy detection of intrachromosomal imbalances without need of metaphase preparations, detects malignant cells and can be extended to other carcinomas for which chromosome 1 arm imbalances are recurrent or chromosome alterations specific of other malignancies. In comparison to other interphase fluorescence in situ hybridization techniques, it avoids every spot scoring problem encountered when using centromeric probes and the difficulties in interpreting structural rearrangements.

Adult↗
PubMed1997

Signal amplification of FISH for automated detection using image cytometry.

The purpose of this study was to improve the detection of FISH signals, in order that spot counting by a fully automated image cytometer be comparable to that obtained visually under the microscope. Two systems of spot scoring, visual and automated counting, were investigated in parallel on stimulated human lymphocytes with FISH using a biotinylated centromeric probe for chromosome 3. Signal characteristics were first analyzed on images recorded with a coupled charge device (CCD) camera. Number of spots per nucleus were scored visually on these recorded images versus automatically with a DISCOVERY image analyzer. Several fluochromes, amplification and pretreatments were tested. Our results for both visual and automated scoring show that the tyramide amplification system (TSA) gives the best amplification of signal if pepsin treatment is applied prior to FISH. Accuracy of the automated scoring, however, remained low (58% of nuclei containing two spots) compared to the visual scoring because of the high intranuclear variation between FISH spots.

Chromosomes, Human, Pair 3↗
PubMed1985

Cleavage within an RNase III site can control mRNA stability and protein synthesis in vivo.

We report that processing at a cloned bacteriophage T7 RNase III site results in strong stabilization of the mRNA relative to the full-length transcript. In contrast, processing by RNase III of the bacteriophage lambda int transcript leads to rapid degradation of the messenger. It is proposed that the mode of cleavage within the RNase III site determines mRNA stability. Single cleavage leaves part of the phage T7 RNase III site in a folded structure at the generated 3' end and stabilizes the upstream mRNA whereas double cleavage at the lambda int site removes the folded structure and accelerates degradation. In addition, the processed transcript is as active a messenger as the unprocessed one and can direct protein synthesis for longer times. This increased efficiency is accompanied by a proportional (3-4 fold) increase in protein levels. In contrast, processing at the lambda int site reduces Int synthesis. Thus, processing may either stabilize mRNA and stimulate gene expression or destabilize a messenger and prevent protein synthesis. The end result appears to be determined by the mode of cleavage within the RNase III site.

DNA Restriction Enzymes↗
PubMed1981

Specific deletion of DNA sequences between preselected bases.

Blunt-end ligation of a "filled-in" HindIII, Sal I, Ava I or Bcl I restriction site with a DNA fragment having A, G, C, or T as the terminal 3' nucleotide regenerates the corresponding restriction site. A combination of this property with the action of BAL 31 nuclease which progressively removes base-pairs from the ends of linear DNA, can generate deletions extending to desired pre-selected nucleotides, and introduces unique restriction sites at those positions. Similarly other restriction sites can be used to select for the deletion of sequences between specific di-, tri-, tetra- and penta-nucleotides. Using this method, 10 base pairs were deleted from the end of a restriction fragment carrying the late promoter for bacteriophage T7 gene 1.1, to create a molecule with a unique restriction site at the initiation codon for translation.

Base Sequence↗
PubMedDate not supplied

Regulation of trophoblastic gelatinases by proto-oncogenes.

During the first trimester of pregnancy, certain cytotrophoblastic cells (CTB) of anchoring villi invade the underlying decidua. Regulation of this invasive behaviour depends on cytokines and growth factors secreted by decidua and trophoblast, which modulate metalloproteinase (MMP) secretion of CTB. Since MMP-9 expression by CTB is a prerequisite for matrigel invasion and since the promoter region of the MMP-9 gene contains two AP-1 binding sites, we hypothesized, that transient activation of c-jun and c-fos oncogenes (which bind to form AP-1) by tumour necrosis factor (TNFalpha), or the phorbol ester TPA will promote the invasive phenotype of CTB and induce the production of MMP-9.TNFalpha or TPA when added to primary cultures of CTB increase MMP-9 activity and MMP-9 mRNA. This effect is inhibited by cycloheximide indicating the necessity of protein synthesis. TPA or TNFalpha induces also the binding of nuclear proteins (extracted from treated CTB) to a radiolabelled oligonucleotide corresponding to the consensus sequence of the TPA responsive element. Antibodies to Jun and Fos can displace this binding. Transient transfection of antisense mRNA to jun or fos into CTB inhibits the immunoreactivity and gelatinolytic activity of MMP-9. We conclude that AP-1 is necessary but may not be sufficient for transactivation of the MMP-9 gene in human CTB.

Adult↗