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At least 649 records · Page 36Linked to original sources
PubMed2002

Distribution of the intermediate filament nestin in the muscularis propria of the human gastrointestinal tract.

The intermediate filament nestin is expressed in neural stem cells, neuroectodermal tumors and various adult tissues. In the gastrointestinal (GI) tract, nestin has been reported in glial cells. Recently, nestin has been reported in interstitial cells of Cajal (ICC) and in gastrointestinal stromal tumors, thought to derive from ICC. Here we investigated nestin immunoreactivity (-ir) in the normal human GI tract, with emphasis on Kit-ir ICC. Two different antibodies specific for human nestin and multicolor high-resolution confocal microscopy were used on material from our human GI tissue collection. The staining pattern of both nestin antibodies was similar. In labeled cells, nestin-ir appeared filamentous. Most intramuscular ICC in antrum and all myenteric ICC (ICC-MP) in small intestine were nestin-ir, while nestin-ir was not detected in deep muscular plexus ICC. In the colon, some - but not all - ICC-MP and most ICC in the circular musculature were nestin-ir while nestin-ir was not detected in ICC in the longitudinal musculature and in the submuscular plexus. In addition, many Kit-negative cells were nestin-ir in all regions. Neurons and smooth muscle cells were consistently nestin negative, while most S100-ir glial cells were nestin-ir. In addition, nestin-ir was also present in some CD34-ir fibroblast-like cells, in endothelium and in other cell types in the mucosa and serosa. In conclusion, nestin-ir is abundantly present in the normal human GI tract. Among a number of cell types, several, but not all, subpopulations of Kit-ir ICC were nestin-ir. The functional significance of nestin in the GI tract remains obscure.

Adolescent↗
PubMed2006

Hyperinduction of nitrilases in filamentous fungi.

2-Cyanopyridine proved to act as a powerful nitrilase inducer in Aspergillus niger K10, Fusarium solani O1, Fusarium oxysporum CCF 1414, Fusarium oxysporum CCF 483 and Penicillium multicolor CCF 2244. Valeronitrile also enhanced the nitrilase activity in most of the strains. The highest nitrilase activities were produced by fungi cultivated in a Czapek-Dox medium with both 2-cyanopyridine and valeronitrile. The specific nitrilase activities of these cultures were two to three orders of magnitude higher than those of cultures grown on other nitriles such as 3-cyanopyridine or 4-cyanopyridine.

Aminohydrolases↗
PubMed2006

Engineering luminescent quantum dots for in vivo molecular and cellular imaging.

Semiconductor quantum dots are luminescent nanoparticles that are under intensive development for use as a new class of optical imaging contrast agents. Their novel properties such as optical tunability, improved photostability, and multicolor light emission have opened new opportunities for imaging living cells and in vivo animal models at unprecedented sensitivity and spatial resolution. Combined with biomolecular engineering strategies for tailoring the particle surfaces at the molecular level, bio-conjugated quantum dot probes are well suited for imaging single-molecule dynamics in living cells, for monitoring protein-protein interactions within specific intracellular locations, and for detecting diseased sites and organs in deep tissue. In this article, we describe the engineering principles for preparing high-quality quantum dots and for conjugating the dots to biomolecular ligands. We also discuss recent advances in using quantum dots for in vivo molecular and cellular imaging.

Animals↗
PubMed2006

Simultaneous two-color spectral fluorescence lymphangiography with near infrared quantum dots to map two lymphatic flows from the breast and the upper extremity.

Due to their small size and poor access, the lymphatic function has been difficult to study in vivo. Especially difficult is the mapping of lymphatic drainage from two basins into the same node. Quantum dots can be used to perform multicolor images with high fluorescent intensity and are of a nano-size size suitable for lymphatic imaging via direct interstitial injection. Here we show simultaneous two-color in vivo wavelength-resolved spectral fluorescence lymphangiography using two near infrared quantum dots with different emission spectra, which allow non-invasive and simultaneous visualization of two separate lymphatic flows draining the breast and the upper extremity and variations in the drainage patterns and the water sheds within the axillary node. Two-color spectral fluorescence lymphangiography can provide insight into mechanisms of drainage from different lymphatic basins that may lead to sentinel lymph nodes detection of the breast cancer as well as prevention of complications such as lymphedema of the arm.

Animals↗
PubMed2006

Establishment, growth and in vivo differentiation of a new clonal human cell line, EM-G3, derived from breast cancer progenitors.

A new clonal cell line, EM-G3, was derived from a primary lesion of human infiltrating ductal breast carcinoma. The line consisted of cuboidal cells with occasional appearance of more differentiated branched cells apparently involved in cell-to-cell communication. The EM-G3 cells, population doubling time 34 h, are dependent on the epidermal growth factor. Multicolor fluorescence in situ hybridization (mFISH) analysis demonstrated a stable diploid genome with several genetic changes. Immunocytochemical analysis of EM-G3 in vitro revealed positivity for keratins (K) K5, K14, K18, nuclear protein p63, epithelial membrane antigen (EMA) and other proteins indicative of a pattern of mammary epithelium bipotent progenitors. Detection of integrins alpha-6, beta-1, and protein CD44 by cDNA array also pointed to the character of basal/stem cells. In contrast, dominant cells in the human original tumor showed the luminal character (K18+, K19+, K5-, K14-, and p63-). However, cells with the immunocytochemical profile similar to that of cultured EM-G3 cells were found in minor clusters in the patient's tumor sections. The EM-G3 cells formed limited tumors in nu/nu mice. The cells in mouse tumors were organized in primitive ductal-like structures consisting of 1-3 large central luminal-like cells (EMA+) surrounded by peripheral myoepithelial-like cells (p63+/EMA-). The large central cells gradually disintegrated, forming a pseudolumen. Apparently, EM-G3 cells are able to partially differentiate in vivo as well as in vitro. Our results indicate that EM-G3 cells were derived from a premalignant population of common progenitors of luminal and myoepithelial cells that were immortalized in an early stage of tumorigenesis.

Animals↗
PubMed2004

Spontaneous mucosal lymphocyte cytokine profiles in children with autism and gastrointestinal symptoms: mucosal immune activation and reduced counter regulatory interleukin-10.

A lymphocytic enterocolitis has been reported in a cohort of children with autistic spectrum disorder (ASD) and gastrointestinal (GI) symptoms. This study tested the hypothesis that dysregulated intestinal mucosal immunity with enhanced pro-inflammatory cytokine production is present in these ASD children. Comparison was made with developmentally normal children with, and without, mucosal inflammation. Duodenal and colonic biopsies were obtained from 21 ASD children, and 65 developmentally normal paediatric controls, of which 38 had signs of histological inflammation. Detection of CD3+ lymphocyte staining for spontaneous intracellular TNFalpha, IL-2, IL-4, IFNgamma, and IL-10, was performed by multicolor flow cytometry. Duodenal and colonic mucosal CD3+ lymphocyte counts were elevated in ASD children compared with noninflamed controls (p<0.03). In the duodenum, the proportion of lamina propria (LP) and epithelial CD3(+)TNFalpha+ cells in ASD children was significantly greater compared with noninflamed controls (p<0.002) but not coeliac disease controls. In addition, LP and epithelial CD3(+)IL-2+ and CD3(+)IFNgamma+, and epithelial CD3(+)IL-4+ cells were more numerous in ASD children than in noninflamed controls (p<0.04). In contrast, CD3(+)IL-10+ cells were fewer in ASD children than in noninflamed controls (p<0.05). In the colon, LP CD3(+)TNFalpha+ and CD3(+)IFNgamma+ were more frequent in ASD children than in noninflamed controls (p<0.01). In contrast with Crohn's disease and non-Crohn's colitis, LP and epithelial CD3(+)IL-10+ cells were fewer in ASD children than in nondisease controls (p<0.01). There was a significantly greater proportion of CD3(+)TNFalpha+ cells in colonic mucosa in those ASD children who had no dietary exclusion compared with those on a gluten and/or casein free diet (p<0.05). There is a consistent profile of CD3+ lymphocyte cytokines in the small and large intestinal mucosa of these ASD children, involving increased pro-inflammatory and decreased regulatory activities. The data provide further evidence of a diffuse mucosal immunopathology in some ASD children and the potential for benefit of dietary and immunomodulatory therapies.

Adolescent↗
PubMed2005

Methyl salicylate, a soybean aphid-induced plant volatile attractive to the predator Coccinella septempunctata.

Induced volatiles provide a signal to foraging predatory insects about the location of their prey. In Iowa, early in the growing season of soybean, Glycine max, many predacious seven-spotted lady beetles, Coccinella septempunctata, were observed on plants with heavy infestations of soybean aphid, Aphis glycines. We studied whether the attraction of this beetle is caused by the release of specific volatile compounds of soybean plants infested by aphids. Volatile compounds emitted by soybean plants infested by aphids were compared with those of undamaged, uninfested, and artificially damaged plants. Gas chromatography-mass spectrometry analyses revealed consistent differences in the profiles of volatile compounds between aphid-infested soybean plants and undamaged ones. Significantly more methyl salicylate was released from infested plants at both the V1 and V2 plant growth stages. However, release patterns of two other induced plant volatiles, (D)-limonene and (E,E)-alpha-farnesene, differed between the two plant growth stages. Gas chromatographic-electroantennographic detection of volatile extracts from infested soybean plants showed that methyl salicylate elicited significant electrophysiological responses in C. septempunctata. In field tests, traps baited with methyl salicylate were highly attractive to adult C. septempunctata, whereas 2-phenylethanol was most attractive to the lacewing Chrysoperla carnea and syrphid flies. Another common lady beetle, the multicolored Asian lady beetle, Harmonia axyridis, showed no preference for the compounds. These results indicate that C. septempunctata may use methyl salicylate as the olfactory cue for prey location. We also tested the attractiveness of some selected soybean volatiles to alate soybean aphids in the field, and results showed that traps baited with benzaldehyde caught significantly higher numbers of aphids.

Animals↗
PubMed2005

The fluorescence bioassay platforms on quantum dots nanoparticles.

In this paper, we present the optical properties and the platforms on fluorescent quantum dots for biological labeling, biomedical engineering and biosensor in molecular imaging. Quantum dots possess several properties that make them very attractive for fluorescent tagging: broad excitation spectrum, narrow emission spectrum, precise tunability of their emission peak, longer fluorescence lifetime than organic fluorophores and negligible photobleaching. We describe how to take such advantages of quantum dots to develop the technology and employ it to build assay platforms. Finally, ultrasensitivity, multicolor, and multiplexing of the technology of semiconductor quantum dots open up promising and interesting possibilities for bioassay platform.

Biotechnology↗
PubMed2005

Spectral and kinetic analysis of the energy coupling in the PS I-LHC I supercomplex from the green alga Chlamydomonas reinhardtii at 77 K.

Energy transfer processes in the chlorophyll antenna of the PS I-LHCI supercomplexes from the green alga Chlamydomonas reinhardtii have been studied at 77 K using transient absorption spectroscopy with multicolor excitation in the 640-670 nm region. Comparison of the kinetic data obtained at low and room temperatures indicates that the slow approximately approximately 100 ps excitation equilibration phase that is characteristic of energy coupling of the LHCI peripheral antenna to the PS I core at physiological temperatures (Melkozernov AN, Kargul J, Lin S, Barber J and Blankenship RE (2004) J Phys Chem B 108: 10547-10555) is not observed in the excitation dynamics of the PS I-LHCI supercomplex at 77 K. This suggests that at low temperatures the peripheral antenna is energetically uncoupled from the PS I core antenna. Under these conditions the observed kinetic phases on the time scales from subpicoseconds to tens of picoseconds represent the superposition of the processes occurring independently in the PS I core antenna and the Chl a/b containing LHCI antenna. In the PS I-LHCI supercomplex with two uncoupled antennas the excitation is channeled to the excitation sinks formed at low temperature by clusters of red pigments. A better spectral resolution of the transient absorption spectra at 77 K results in detection of two DeltaA bands originating from the rise of photobleaching on the picosecond time scale of two clearly distinguished pools of low energy absorbing Chls in the PS I-LHCI supercomplex. The first pool of low energy pigments absorbing at 687 nm is likely to originate from the red pigments in the LHCI where the Lhca1 protein is most abundant. The second pool at 697 nm is suggested to result either from the structural interaction of the LHCI and the PS I core or from other Lhca proteins in the antenna. The kinetic data are discussed based on recent structural models of the PS I-LHCI. It is proposed that the uncoupling of pigment pools may be a control mechanism that regulates energy flow in Photosystem I.

Animals↗
PubMed1992

Fluorescent rhodol derivatives: versatile, photostable labels and tracers.

A series of chemically reactive, fluorescent rhodol derivatives was prepared and evaluated. Reactive functional groups included activated esters, amines, haloacetamides, fixable hydrazide derivatives, acrylamides, and photoaffinity reagents. Depending on the choice of substituents, absorption maxima of the dyes varied from 490 to 550 nm with extinction coefficients that were generally greater than 50,000 M-1 cm-1 in aqueous solution and emission maxima from 520 to 580 nm. Most of the compounds investigated exhibited fluorescence lifetimes between 3 and 4 ns. Individual derivatives were suitable for excitation with the 488 and 514-nm lines of the argon ion laser and the 546-nm line of the mercury arc lamp and were compatible for use with standard fluorescein and rhodamine filter sets. The rhodol dyes were more photostable and less sensitive to pH changes in the physiological range than fluorescein derivatives. Some examples show absorption maxima at or near 514 nm, an excitation wavelength that is useful for multicolor fluorescence microscopy, flow cytometry, and DNA sequencing. Derivatives were also prepared that exhibit absorption and emission maxima similar to those of tetramethylrhodamine (TMR) analogs but with higher quantum yields in aqueous solution. A number of the dyes had higher solubilities in aqueous systems and were less quenched on conjugation to proteins than TMR derivatives. Appropriate substitution results in a wider range of solubilities in hydrophilic or lipophilic solvents than is easily accomplished with fluorescein or TMR derivatives. Conjugates of a number of the rhodol fluorophores were generally more photostable and less pH sensitive than fluorescein conjugates and more fluorescent than TMR conjugates.

Affinity Labels↗
PubMed1992

Multiple nucleic acid labeling and rainbow detection.

A method which allows discrete nucleic acid sequences to be detected with differently colored hybridization signals on the same blot involving only a single hybridization step is described. Nucleic acid probes labeled with digoxigenin, fluorescein, or biotin are hybridized simultaneously to immobilized target nucleic acids. Differential colorimetric detection is carried out in consecutive alkaline phosphatase-based immunoassays with one of three 3-hydroxy-2-naphthoic acid anilide phosphate/diazonium salt combinations as substrate. Each label is visualized by a different color precipitate (green, red, and blue) directly on the membrane. We demonstrate the use of this method in multicolor plasmid mapping, detection of different genomic sequences on a single Southern blot, discrimination of transcription levels in a Northern blot, and colony screening. Advantages and limitations of the method, as well as further applications, are discussed.

Alkaline Phosphatase↗
PubMed1993

Preparation, isolation, and characterization of novel heterogeneous branched cyclomalto-oligosaccharides having beta-D-galactosyl residue(s) on the side chain.

Transgalactosylated products of branched cyclodextrins (glucosyl-alpha CD, -beta CD, -gamma CD, and maltosyl-alpha CD, -beta CD, -gamma CD) were synthesized by beta-D-galactosidases from Bacillus circulans and Penicillium multicolor using lactose as a donor substrate and branched CDs as acceptors. Eighteen beta-D-galactosylated branched CDs were isolated and purified by HPLC. Their structures were elucidated by FABMS and 13C NMR spectroscopies, and methylation analysis. The chromatographic behavior of these novel heterogeneous branched CDs on three HPLC columns of different separation modes was compared.

Bacillus↗
PubMed1990

Single laser flow cytometric detection of lymphocytes binding three antibodies labelled with fluorescein, phycoerythrin and a novel tandem fluorochrome conjugate.

In order to implement three-color surface immunofluorescence on a single laser flow cytometer, we combined a new fluorescent tandem conjugate (TC, R-phycoerythrin plus Texas red, available as DuoCHROME) with fluorescein (FITC)- and phycoerythrin (PE)-labelled monoclonal antibodies for the simultaneous detection of three antigens on individual lymphoid cells. Considerable amounts of fluorescence leaked from the PE component of the TC complex into the PE detector, which necessitated compensation of the PE signal. Single and double stainings of peripheral lymphocytes with FITC-, PE- and/or TC-labelled antibodies revealed that the individual labels all identified populations of similar size, and that subsets of T cells could be properly and accurately detected with TC as one label. Triple staining was used to study phenotypically activated HLA-DR+ cells within functionally important subsets of CD4+ T cells, i.e., CD45R+ CD4+ (naive, 'suppressor inducer' T) and UCHL1+ CD4+ (memory, 'helper' T) cells. In the blood of healthy subjects as well as patients with primary biliary cirrhosis, CD4+ UCHL1+ (i.e., memory) T cells were significantly more activated than CD4+ UCHL1- cells. The reciprocal CD45R+ CD4+ (naive) T subset expressed HLA-DR to a much lesser extent. In patient samples, this difference in CD4+ T subset activation was more pronounced than among normal lymphocytes. The described triple immunofluorescence staining technique facilitates true multicolor analysis of individual cells on a single laser flow cytometer. Such multiparameter investigations may prove to be important for the further understanding of the phenotypic and functional diversity of immunocompetent cells.

Antigens, Differentiation↗
PubMed1995

Flow cytometric determination of apoptosis in heterogeneous cell populations.

We have shown that apoptosis and surface antigen expression can be detected simultaneously using multicolor flow cytometry. Apoptosis was measured with an in situ assay that makes use of the ability of the enzyme terminal deoxyribonucleotidyl transferase to catalyze the addition of biotinylated nucleotides to the free 3'-OH groups produced during endonucleolytic cleavage of DNA. The incorporation of the biotinylated nucleotides was quantified using fluorochrome-coupled streptavidin. Immunofluorescence was used to identify the phenotype of apoptotic cells using three color flow cytometry. In model systems, apoptosis was detected in human PBMNC treated with the DNA topoisomerase I inhibitor CAM and in murine thymocytes treated with DEX. The simultaneous application of this method for detecting apoptosis and immunofluorescence offers several advantages. (1) Apoptotic and necrotic cells can be distinguished. (2) The phenotype of the cells undergoing apoptosis can be determined in heterogeneous systems. Thus, we could show that T cells are relatively resistant to CAM-induced apoptosis compared to other peripheral blood mononuclear cells and that both double negative and single positive thymocytes are resistant to steroid-induced apoptosis. (3) The flow cytometric TdT assay to detect apoptosis-associated DNA degradation method is extremely sensitive compared with gel electrophoresis. As few as 2-5 x 10(3) cells give an adequate signal and it is possible to detect DNA strand breaks even when only a small proportion of the cells are undergoing apoptosis. Neither the sensitivity nor specificity of this method can be matched by any electrophoretic method of detecting apoptosis.

Animals↗
PubMed1995

Isolation of leukocytes infiltrating the islets of Langerhans of diabetes-prone mice for flow cytometric analysis.

A simple method was developed for flow cytometric immunofluorescence analysis of cells infiltrating the islets of Langerhans in diabetes-prone rodents. Pancreases were submitted to collagenase P digestion and, in order to minimize collagenase action on leukocytes, islets were isolated before digestion was completed, that is, when exocrine tissue still remained around the islets. Islets, maintained in medium supplemented with sodium azide to prevent modulation of surface markers, were then pressed through a metal sieve and the cell suspension filtered through two different nylon screens. Cells were washed before immunofluorescence staining. Comparative phenotyping of spleen cells submitted to a similar treatment showed that, provided the collagenase P batch was screened, this procedure did not alter leukocyte surface markers and allowed multicolor analysis of the islet infiltrating cells.

Animals↗
PubMed1987

Rift Valley fever virus transmission by different Egyptian mosquito species.

4 Egyptian mosquito species were tested for their ability to transmit the Egyptian ZH-501 strain of Rift Valley fever virus (RVFV) to golden Syrian hamsters. Culex (Cx.) antennatus was the most efficient vector, showing a 37.5% transmission rate following a hamster blood meal containing 10 suckling mouse intracerebral 50% lethal doses (SMILD50) per ml. Fully engorged mosquitoes of this species showed an infection rate of 85% with the mean viral titres of transmitting mosquitoes 100-fold higher than non-transmitters. Autogenous and anautogenous populations of Aedes (Ae.) caspius were tested separately, and the transmission rates were 23.1% and 9.7% respectively, following feeding on hamsters with similar levels of viraemia. Two anopheline species, Anopheles (An.) multicolor and An. pharoensis, showed 12.5% and 3.5% transmission rates under similar conditions. In these 3 species infection rates exceeded 75% and mosquitoes transmitting had a higher average titre than those not transmitting.

Animals↗
PubMed1993

Picogram detection of stable dye-DNA intercalation complexes with two-color laser-excited confocal fluorescence gel scanner.

The stable complexes between highly fluorescent, polyfunctional intercalators and dsDNA can be used to detect dsDNA in agarose gels at picogram levels and for multicolor detection of multiplexed dsDNA fragments. Development of additional DNA-binding fluorophores with appropriate spectroscopic properties will expand the range of applications. In principle, the DNA-dye intercalation complexes represent a more sensitive alternative to an established approach to fluorescent labeling and detection of restriction fragments by ligation to single-stranded short oligonucleotides labeled with different fluorochromes, followed by separation on denaturing polyacrylamide gels. The latter technique gives near single-base resolution up to 400 bases and the ability to quantitate fragment size up to 2000 bases, and has been successfully applied to cosmid mapping. Detection of DNA fragments as intercalation complexes requires that the separations be performed on agarose gels under nondenaturing conditions. Such conditions have been used for extensive mapping of yeast cosmids with postelectrophoresis staining with ethidium bromide. For the patterns on agarose gels, the magnitude of the "error window," which specifies how similar two fragments must be before the corresponding fragments in different digests are paired, was reported to be strongly size dependent. The error window was expanded by a factor of 1.3 for fragments from 400 to 600 bp, 1.2 for fragments from 600 to 800 bp, and 1.1 for fragments from 800 to 1000 bp. Moreover, it was necessary to introduce corrections for systematic differences between size estimates taken from two different gels. For the multiplexing procedure described here, the size estimates for fragments from 600 bp to over 23 kbp were in close agreement with actual sizes as determined from DNA sequence (Table I), and certainly within the error windows given above. The multiplexing procedure should also minimize errors introduced by gel-to-gel variations in mobility, because the standard and unknowns are always run in the same lanes. Kohara et al. established a physical map of almost the entire Escherichia coli chromosome by analysis of a large genomic library. In this case, partial restriction digests were used to generate patterns of fragments and the mapping was performed by agarose gel electrophoresis. The disadvantage of this approach is that fewer fragments are generated. However, this is compensated for by the fact that partial digests reveal the order of the fragments produced and thus greatly increase the amount of information relevant to the question of overlap between different DNA fragments.(ABSTRACT TRUNCATED AT 400 WORDS)

Benzothiazoles↗
PubMed1993

Immunoglobulin heavy and light chain genes rearrange independently at early stages of B cell development.

The compartment of mouse B cell progenitors can be resolved into five developmentally related fractions by multicolor flow cytometry. Using this system and employing mutant mice in which the membrane exon of the mu chain, the lambda 5 gene, or the JH locus was inactivated by gene targeting, we found that expression of the pre-B cell receptor complex is necessary for the transition from the large CD43+ to the small CD43- pre-B cell stage. We report the occurrence of immunoglobulin heavy and light chain gene rearrangement at the stage of large B cell precursors. We show that neither the pre-B cell receptor complex nor any gene rearrangement in the heavy chain locus is required for the induction of kappa light chain gene rearrangement in early B cell progenitors.

Animals↗