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Biomedical subjects

C Nguyen

Publications and source records attributed to C Nguyen.

At least 73 records · Page 4Linked to original sources
PubMed1999

Increased peripheral nerve microvessels in early experimental diabetic neuropathy: quantitative studies of nerve and dorsal root ganglia.

Microangiopathy is an important complication of diabetes mellitus and neovascularity is a feature of human diabetic retinopathy. The objective of this work was to evaluate numbers, areas and size distributions of whole nerve, endoneurial and dorsal root ganglia perfused microvessels in a detailed fashion using unfixed tissues from rats with experimental diabetes. Experimental neuropathy was studied in male Sprague-Dawley rats 12 weeks after streptozotocin or citrate buffer injection. Electrophysiological recordings of sciatic-tibial motor and caudal sensory fibers identified conduction slowing in diabetes indicating neuropathy. Diabetics had a rise in the numbers of whole nerve microvessels and endoneurial microvessels with associated rises in vessel densities and total vessel luminal areas. Increased vessel numbers in 15-30 microm diameter size ranges were particularly prominent. There was a rise in summed vascular areas in diabetics but the mean luminal area of vessels was not increased. Similar, but not significant trends were observed in a selective analysis of endoneurial vessels alone. In contrast, dorsal root ganglia microvessels were not increased in number. Early experimental diabetic neuropathy is associated with increased numbers of microvessels supplying the peripheral nerve trunk, likely representing neovascularity.

Animals↗
PubMed1999

Differential expression assay of chromosome arm 8p genes identifies Frizzled-related (FRP1/FRZB) and Fibroblast Growth Factor Receptor 1 (FGFR1) as candidate breast cancer genes.

Deletions and amplifications are frequent alterations of the short arm of chromosome 8 associated with various types of cancers, including breast cancers. This indicates the likely presence of tumor suppressor genes and oncogenes. In the present study, we have used the expressed sequence tag (EST) map of 8p11-21 to assemble a set of available cDNAs representing genes from this region. DNA arrays were prepared for expression analysis and search for genes potentially involved in breast cancer. Underexpresion in tumoral breast cells (versus normal breast) was observed for 15 transcripts. Among these, the Frizzled-related gene FRP1/FRZB, was turned off in 78% of breast carcinomas, suggesting that the lack of its product may be associated with malignant transformation. Overexpression in tumoral breast cells was observed for 13 genes. The FGFR1 gene, that encodes a tyrosine kinase receptor for members of the fibroblast growth factor family, was identified as a good candidate for one amplification unit. Taken together, our results demonstrate that such a strategy can rapidly identify genes with an altered pattern of expression and provide candidate genes for malignancies.

Breast Neoplasms↗
PubMed1999

CT and PET lung image registration and fusion in radiotherapy treatment planning using the chamfer-matching method.

PURPOSE: We present a validation study of CT and PET lung image registration and fusion based on the chamfer-matching method. METHODS AND MATERIALS: The contours of the lung surfaces from CT and PET transmission images were automatically segmented by the thresholding technique. The chamfer-matching technique was then used to register the extracted lung surfaces. Arithmetic means of distance between the two data sets of the pleural surfaces were used as the cost function. Matching was then achieved by iteratively minimizing the cost function through three-dimensional (3D) translation and rotation with an optimization method. RESULTS: Both anatomic thoracic phantom images and clinical patient images were used to evaluate the performance of our registration system. Quantitative analysis from five patients indicates that the registration error in translation was 2-3 mm in the transverse plane, 3-4 mm in the longitudinal direction, and about 1.5 degree in rotation. Typical computing time for chamfer matching is about 1 min. The total time required to register a set of CT and PET lung images, including contour extraction, was generally less than 30 min. CONCLUSION: We have implemented and validated the chamfer-matching method for CT and PET lung image registration and fusion. Our preliminary results show that the chamfer-matching method for CT and PET images in the lung area is feasible. The described registration system has been used to facilitate target definition and treatment planning in radiotherapy.

Aged↗
PubMed1999

'LABNOTE', a laboratory notebook system designed for academic genomics groups.

We have developed a relational laboratory database system, adapted to the daily book-keeping needs of laboratories that must keep track of information acquired on hundreds or thousands of clones in an effective and user-friendly fashion. Data, whether final or related to experiments in progress, can be accessed in many different ways, e.g. by clone name, by gene, by experiment or through DNA sequence. Updating, import and export of results is made easier by specially developed tools. This system, in network version, serves several groups in our Institute and (over the Internet) elsewhere, and is instrumental in collaborative studies based on expression profiling. It can be used in many similar situations involving progressiveaccumulation of information on sets of clones or related objects.

Database Management Systems↗
PubMed1999

Differential gene expression in CD3epsilon- and RAG1-deficient thymuses: definition of a set of genes potentially involved in thymocyte maturation.

A set of 3000 mouse thymus cDNAs was analyzed by extensive measurement of expression using complex-probe hybridization of DNA arrays ("quantitative differential screening"). The complex probes were initially prepared using total thymus RNA isolated from C57BL/6 wild-type (WT), CD3epsilon- and RAG1-deficient mice. Over 100 clones displaying over- or under-expression by at least a factor of two between WT and knockout (KO) thymuses were further analyzed by measuring hybridization signatures with probes from a wide range of KO thymuses, cell types, organs, and embryonic thymuses. A restricted set of clones was selected by virtue of their expression spectra (modulation in KO thymuses and thymocytes, lymphoid cell specificity, and differential expression during embryonic thymus development), sequenced at one extremity, and compared to sequences in databases. Clones corresponding to previously identified genes (e.g., Tcrbeta, Tcf1 or CD25) showed expression patterns that were consistent with existing data. Ten distinct clones corresponding to new genes were subjected to further study: Northern blot hybridization, in situ hybridization on thymus sections, and partial or complete mRNA sequence determination. Among these genes, we report a new serine peptidase highly expressed in cortical epithelial cells that we have named thymus-specific serine peptidase (TSSP), and an acidic protein expressed in thymocytes and of unknown function that we have named thymus-expressed acidic protein (TEAP). This approach identifies new molecules likely to be involved in thymocyte differentiation and function.

Alternative Splicing↗
PubMed1999

Occult cervical metastases: immunoperoxidase analysis of the pathologically negative neck.

OBJECTIVES: The purpose of this study is to better estimate the true incidence of occult regional metastases associated with stage I and II squamous cell carcinoma of the oral cavity. The clinical and prognostic significance of micrometastatic disease discovered by cytokeratin immunoperoxidase reactivity in the previously pathologically N0 neck is also evaluated. METHODS: Forty patients treated between 1985 and 1996 with T1 or T2 squamous cell carcinoma of the lip and oral cavity were studied. All had primary surgical treatment including functional neck dissection. No metastases were demonstrated on hematoxylin and eosin microscopy. All specimens were reexamined with immunoperoxidase staining for cytokeratin. RESULTS: Five percent of patients had micrometastatic disease. Retrospective analysis of patients with a minimum follow-up of 2 years has failed to show a statistically significant association between a positive cytokeratin analysis and poor locoregional control or overall survival. CONCLUSIONS: Results suggest that the true incidence of occult metastases with carcinoma of the oral cavity is significantly higher than previously documented. However, the prognostic significance of these findings remains unclear.

Adult↗
PubMed1999

Sensitivity issues in DNA array-based expression measurements and performance of nylon microarrays for small samples.

DNA or oligonucleotide arrays are widely used for large-scale expression measurements, using various implementations: macroarrays in which DNA is spotted onto nylon membranes of relatively large dimensions (with radioactive detection) on the one hand; microarrays on glass slides and oligonucleotide chips, both used with fluorescent probes, on the other hand. Nylon micro-arrays with colourimetric detection have also been described recently. The small physical dimensions of miniaturized systems allow small hybridization volumes (2-100 microl) and provide high probe concentrations, in contrast to macroarrays. We show, however, that actual sensitivity (defined as the amount of sample necessary for detection of a given mRNA species) is in fact similar for all these systems and that this is mostly due to the very different amounts of target material present on the respective arrays. We then demonstrate that the combination of nylon microarrays with(33)P-labelled radioactive probes provides 100-fold better sensitivity, making it possible to perform expression profiling experiments using submicrogram amounts of unamplified total RNA from small biological samples. This has important implications in basic and clinical research and makes this alternative approach particularly suitable for groups operating in an academic context.

Biosensing Techniques↗
PubMed1999

Preclinical pharmacokinetics, interspecies scaling, and tissue distribution of a humanized monoclonal antibody against vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) plays a crucial role in angiogenesis and in pathological processes such as tumor growth, rheumatoid arthritis, and ocular neovascularization. A recombinant humanized monoclonal antibody (rhuMAb), rhuMAb VEGF, has been developed to inhibit the effects of VEGF in the treatment of solid tumors. Intravenous and s.c. pharmacokinetic studies were conducted in mice, rats, and cynomolgus monkeys. In addition, the tissue distribution of i.v. 125I-rhuMAb VEGF was investigated in rabbits. At a dose of approximately 10 mg/kg, the clearance of rhuMAb VEGF from the serum was 15.7 ml/day/kg in mice, 4.83 ml/day/kg in rats, and 5.59 ml/day/kg in cynomolgus monkeys, and the terminal half-life ranged from 6 to 12 days in all species. After s.c. administration, rhuMAb VEGF had a bioavailability of 69% in rats and 100% in mice and cynomolgus monkeys. Pharmacokinetic data in mice, rats, and cynomolgus monkeys were used to predict the pharmacokinetics of rhuMAb VEGF using allometric scaling in humans. The predicted serum clearance of rhuMAb VEGF in humans was 2.4 ml/day/kg and the terminal half-life was 12 days. Two hours after i.v. bolus administration of 125I-rhuMAb VEGF in rabbits, trichloroacetic acid-precipitable radioactivity was noted primarily in the plasma, with lesser amounts in highly perfused tissues such as kidneys, testes, spleen, heart, and lungs. At 48 h after dosing, trichloroacetic acid-precipitable radioactivity was noted in plasma with minimal distribution to testes, bladder, heart, lungs, and kidneys. Tissue distribution and pharmacokinetic data indicate that rhuMAb VEGF is cleared slowly and distributes to specific sites in the body.

Animals↗
PubMed1998

Dipeptidyl-peptidase IV-beta--further characterization and comparison to dipeptidyl-peptidase IV activity of CD26.

Dipeptidyl peptidase IV-beta (DPP IV-beta) is a novel protein which shows a peptidase activity similar to the T-cell-activation antigen CD26. To further characterize this DPP IV-beta and confirm its cell surface expression, we have developed a purification strategy using the CD26- cell line C8166. The purification process includes biotinylation of cell surface proteins before preparation of cell extracts and processing by gel-filtration, ion-exchange and lectin chromatographies. Consistent with the molecular mass of DPP IV-beta estimated by gel-filtration chromatography, the final purified fraction, manifesting a typical DPP IV activity, showed a major biotinylated 75-80-kDa band in SDS/PAGE, thus suggesting the monomeric nature of this enzyme. Kinetic parameters of DPP IV-beta and the sensitivity to a new family of irreversible DPP IV inhibitors, were studied in comparison to CD26. Both enzymes followed a Michaelis kinetics with different Km values for Gly-Pro-NH-Np (NH-Np, para-nitroanilide) hydrolysis (0.28+/-0.05 mM and 0.12+/-0.02 mM). More significant differences were observed in the sensitivity to inhibitors, which exerted a much higher activity on CD26 than on DPP IV-beta. These differences permitted us to study DPP IV-beta expression in CD26-expressing cells, showing the expression of this new enzyme in all lymphoid cells tested, and a rapid enhancement in phytohemagglutinin-stimulated or protein-A-stimulated peripheral blood mononuclear cells. Our results indicate that, although DPP IV-beta and CD26 are coexpressed and manifest a typical DPP IV activity, there are distinct features in their catalytic activities that may confer to each enzyme a complementary role in peptide processing.

Biotinylation↗
PubMed1998

Specific and irreversible cyclopeptide inhibitors of dipeptidyl peptidase IV activity of the T-cell activation antigen CD26.

The dipeptidyl peptidase IV (DPP IV) activity of CD26 is characterized by its post-proline-cleaving capacity that plays an important but not yet understood role in biological processes. Here we describe a new family of specific and irreversible inhibitors of this enzyme. Taking into account the substrate specificity of DPP IV for P2-P1><-P1' cleavage, we have designed and synthesized cyclopeptides c[(alphaH2N+)-Lys-Pro-Aba-(6-CH2-S+R2)-Glyn] 2TFA- (Aba = 3-aminobenzoic acid, R = alkyl) possessing a proline at the P1 position and a lysine in the P2 position, which allows the closing of the cycle on its side chain. These molecules show a free N-terminus, necessary for binding to the CD26 catalytic site, and a latent quinoniminium methide electrophile, responsible for inactivation. Treatment of c[alphaZ-Lys-Pro-Aba-(6-CH2-OC6H5)-Glyn], obtained by peptide synthesis in solution, with R2S/TFA simutaneously cleaved the Z protecting group and the phenyl ether function and led to a series of cyclopeptide sulfonium salts. These cyclopeptides inhibited rapidly and irreversibly the DPP IV activity of CD26, with IC50 values in the nanomolar range. Further studies were carried out to investigate the effect of the modification of the ring size (n = 2 or 4) and the nature of the sulfur substituents (R = Me, Bu, Oct). Cycle enlargement improved the inhibitory activity of the methylsulfonio cyclopeptide, whereas the increase of the alkyl chain length on the sulfur atom had no apparent effect. Other aminopeptidases were not inhibited, and a much weaker activity was observed on a novel isoform of DPP IV referred to as DPP IV-beta. Thus, this new family of irreversible inhibitors of DPP IV is highly specific to the peptidase activity of CD26.

Animals↗
PubMed1998

Generation of intestinal mucosal lymphocytes in SCID mice reconstituted with mature, thymus-derived T cells.

Transfer of peripheral lymph node lymphocytes to SCID mice leads to the long term establishment of mucosal T lymphocytes within the epithelium and lamina propria of the small and large intestines. Analysis of engrafted intraepithelial lymphocytes (IEL) showed that they had acquired a surface phenotype that in several respects is typical of IEL. In addition, the functional profile of engrafted IEL derived from lymph node T cells was similar to that of normal IEL; as the donor-derived T cells exhibited a strong cytolytic activity, a poor proliferative response to mitogenic stimuli, and a tendency to home and expand specifically in the intestine upon transfer to secondary SCID recipients. Optimal engraftment of intestinal T cells required bacterial flora, as the number of lymphocytes was greatly reduced in SCID recipients with a reduced flora. These results demonstrate that mature, thymus-derived T cells can migrate to the intestine and become functionally specialized to the intestinal milieu. The acquisition of phenotypic markers characteristic of the intestinal microenvironment by engrafted cells suggests that T cell migration of lymphocytes to the SCID intestine is not aberrant, but it may reflect processes that are ongoing in immunocompetent mice. Furthermore, these data suggest that the homing and/or expansion of typical, thymus-derived T cells in the intestine may be driven by luminal Ags such as those derived from bacterial flora.

Animals↗
PubMed1998

PMA alters folate receptor distribution in the plasma membrane and increases the rate of 5-methyltetrahydrofolate delivery in mature MA104 cells.

MA104 cells (a monkey kidney cell line) can internalize 5-methyltetrahydrofolate via a receptor mediated process termed potocytosis. Uptake is initiated by binding to an external folate receptor which cycles to an internal, but membrane bound compartment. These two pools can be measured by determining the amount of [3H]ligand removed by an acid-saline wash, i.e. acid labile and acid resistant pools. When assayed in confluent nonmitotic cells, 2/3 of the folate receptor pool is located in an internal (acid resistant) compartment, but phorbol 12-myristate 13-acetate (PMA) causes a shift such that 65-75% of the receptor pool resides on the surface of the plasma membrane. This new steady state is likely the result of an increased rate of receptor movement. In addition, PMA increases the rate of 5-methyl[3H]tetrahydrofolate delivery to the cytoplasm 1.8 fold. Using known inhibitors of potocytosis, we were able to show that the increased rate of delivery is receptor mediated. Comparison of the time courses of the PMA effects on folate receptor redistribution assessed by membrane binding of [3H]folic acid and 5-methyl[3H]tetrahydrofolate delivery to the cytoplasm suggests that PMA may be activating more than one protein kinase C independent signal transduction pathway. PMA is the first reported positive modulator of receptor mediated folate uptake.

Animals↗
PubMed1998

The genomic organization of NKG2C, E, F, and D receptor genes in the human natural killer gene complex.

Interactions of natural killer cell receptors with their cognate ligands play a major role in regulating NK cell function. The NKG2 gene family encodes several highly similar proteins, which are known to form heterodimers with the CD94 receptor. These dimers play a role in the inhibition as well as the activation of NK cells. We have analyzed the gene structures of the NKG2C, D, E, and F genes, and determined their genomic organization. Restriction mapping and sequencing revealed the four genes to be closely linked to one another, and of the same transcriptional orientation. An exon duplication within the NKG2C and E genes was identified, although the duplicated version of this exon has not yet been found in mRNA sequences. The NKG2C, E, and F genes, despite being highly similar, are variable at their 3' ends. We show that NKG2C consists of six exons, whereas NKG2E has seven, and the splice acceptor site for the seventh exon occurs in an Alu repeat. NKG2F consists of only four exons and part of exon IV is in some cases spliced to the 5' end of the NKG2D transcript. NKG2D has only a low similarity to the other NKG2 genes.

Base Sequence↗
PubMed1998

The utility of aminoglycosides in an era of emerging drug resistance.

As the problem of global antibiotic resistance continues to worsen, aminoglycosides have assumed increasing importance in clinical practice. Their broad antimicrobial spectrum, rapid bactericidal action, and ability to act synergistically with other drugs have made them especially useful in the treatment of serious nosocomial infections. However, as with other drugs, their overuse and misuse leads to the development of resistance in important microbial pathogens. The appropriate use of the aminoglycosides is essential to assure their continued efficacy. Therefore, physicians must familiarize themselves with both the clinical indications and the limitations of these drugs if they are to remain efficacious in the years to come.

Aminoglycosides↗
PubMed1998

Stable and monodisperse lipoplex formulations for gene delivery.

A stable single vial lipoplex formulation has been developed that can be stored frozen without losing either biological activity or physical stability. This formulation was identified by systematically controlling several formulation variables and without introducing either stabilizers or surfactants. Analytical assays were used to unambiguously characterize the formulations. The critical formulation parameters were: (1) the size of the cationic liposomes; (2) the rate and method of DNA and cationic liposome mixing; and (3) the ionic strength of the suspending vehicle. The mixing conditions were precisely controlled by using a novel, specially designed continuous flow pumping system in which the DNA and liposome solutions were mixed at the junction of a T-connector. Homogenous cationic liposome preparations were prepared by extrusion in two different size ranges of either 400 or 100 nm. Extruded liposomes produced more monodisperse and physically stable lipoplex formulations than unextruded liposomes, but the formulations prepared with 100 nm liposomes were less active in in vitro transfection assays than either the 400 nm or unextruded liposomes. Low ionic strength and 5% sorbitol were required for the lipoplex formulations to survive freezing and thawing. A frozen lipoplex formulation stored for more than a year maintained its biological activity. These results have broad implications for the pharmaceutical development of lipoplex formulations for gene delivery.

Animals↗
PubMed1998

Differential use of transcription activation function 2 domain of the vitamin D receptor by 1,25-dihydroxyvitamin D3 and its A ring-modified analogs.

Analogs of 1,25-dihydroxyvitamin D3 (1,25D3) can be used to elucidate details of vitamin D receptor (VDR) activation. The A ring-modified analog, (TN-2) has 15-fold less affinity for VDR, but its transcriptional activity is diminished 1000-fold. Likewise, the ability of TN-2 to induce a protease-resistant conformation in VDR is 1/1000 that of 1,25D3. The stability of the VDR-TN-2 complexes is also significantly lower than VDR-1,25D3 complexes. Mapping the VDR-binding site of TN-2 showed that it had a significantly greater requirement for transcription activation function 2 (AF-2) residues than 1,25D3 did. These results suggest that the increased requirement for AF-2 residues that was induced by the A ring modifications is associated with diminished receptor activation. To determine whether restoring the potency of TN-2 by additional structural modifications would change the requirements for AF-2 residues, we synthesized hybrid analogs with 1beta-hydroxymethyl-3-epi groups and with dimethyl groups at positions 26 and 27 of the side chain, without or with a double bond between CD ring positions 16 and 17. We found that the side chain modification enhanced transcriptional activity 150-fold, increased the ability of the receptor to form a protease-resistant conformation 100-fold, and stabilized the VDR-analog complexes. The addition of the 16-ene group further reduced the analog's dissociation rate and increased its potency in the protease assays. These functional changes in the hybrid analogs were associated with a significant reduction in interaction with AF-2 residues. We conclude that there is an inverse relationship between analogs' potencies and their interaction with AF-2 residues of VDR.

Animals↗