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1
Voss, H., J. Zimmermann, C. Schwager, H. Erfle, J. Stegemann, K. Stucky, and W. Ansorge. "Automated fluorescent sequencing of lambda DNA." Nucleic Acids Research 18, no. 17 (1990): 5314. http://dx.doi.org/10.1093/nar/18.17.5314.
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Lallement, Pierre-Alexandre, Edgar Meux, José M. Gualberto, Pascalita Prosper, Claude Didierjean, Frederick Saul, Ahmed Haouz, Nicolas Rouhier, and Arnaud Hecker. "Structural and enzymatic insights into Lambda glutathione transferases from Populus trichocarpa, monomeric enzymes constituting an early divergent class specific to terrestrial plants." Biochemical Journal 462, no. 1 (July 24, 2014): 39–52. http://dx.doi.org/10.1042/bj20140390.
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The resolution of Lambda GST crystal structures in complex with glutathione and competition experiments with fluorescent probes reveal that poplar Lambda GSTs are involved in the reduction of glutathionylated substrates.
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Wu, Tongbo, Xianjin Xiao, Feidan Gu, and Meiping Zhao. "Sensitive discrimination of stable mismatched base pairs by an abasic site modified fluorescent probe and lambda exonuclease." Chemical Communications 51, no. 98 (2015): 17402–5. http://dx.doi.org/10.1039/c5cc05749c.
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An abasic site modified fluorescent probe has been developed which enabled the rapid discrimination of stable single mismatched base pairs by lambda exonuclease with remarkably high discrimination factors (447 for T:G and 238 for A:G).
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Chen, F. T., and R. A. Evangelista. "Feasibility studies for simultaneous immunochemical multianalyte drug assay by capillary electrophoresis with laser-induced fluorescence." Clinical Chemistry 40, no. 9 (September 1, 1994): 1819–22. http://dx.doi.org/10.1093/clinchem/40.9.1819.
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Abstract We present a method for the simultaneous quantification of multiple drug analytes in urine, based on combining immunochemical binding with capillary electrophoretic separation. Two fluorescent drug-cyanine (Cy) dye conjugates were prepared as competing species for the immunoassay. Morphine was derivatized with Cy5 (lambda max = 652 nm, epsilon = 215,000 mol-1cm-1 L), phencyclidine (PCP) with Cy5.5 (lambda max = 675 nm, epsilon = 200,000 mol-1cm-1L). The high-efficiency resolving power of the capillary electrophoresis system (20 microns x 27 cm column) separated the individual labeled drugs, and the antigen-antibody complexes were detected by laser-induced fluorescence (laser: 10 mW He-Ne at 632.8 nm) with Cy5 diacid as internal standard. Simultaneous competitive immunoassay of morphine and PCP in urine showed that the free labeled-drug peak areas were proportional to the concentrations of the drug species present in the urine sample. This immunoassay can be performed routinely and reproducibly in < 5 min with analytical detection limits of 4 nmol/L for PCP and 40 nmol/L for morphine.
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Smith, David L., Douglas K. Struck, J. Martin Scholtz, and Ry Young. "Purification and Biochemical Characterization of the Lambda Holin." Journal of Bacteriology 180, no. 9 (May 1, 1998): 2531–40. http://dx.doi.org/10.1128/jb.180.9.2531-2540.1998.
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ABSTRACT Holins are small phage-encoded cytoplasmic membrane proteins, remarkable for their ability to make membranes permeable in a temporally regulated manner. The purification of S105, the λ holin, and one of the two products of gene S is described. Because the wild-type S105 holin could be only partially purified from membrane extracts by ion-exchange chromatography, an oligohistidine tag was added internally to the S105 sequence for use in immobilized metal affinity chromatography. An acceptable site for the tag was found between residues 94 and 95 in the highly charged C-terminal domain of S. This allele, designated S105H94, had normal lysis timing under physiological expression conditions. The S105H94 protein was overproduced, purified, and characterized by circular dichroism spectroscopy, which revealed approximately 40% alpha-helix conformation, consistent with the presence of two transmembrane helices. The purified protein was then used to achieve release of fluorescent dye loaded in liposomes in vitro, whereas protein from an isogenic construct carrying an S mutation known to abolish hole formation was inactive in this assay. These results suggest that S is a bitopic membrane protein capable of forming aqueous holes in bilayers.
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Gillen, C. M., A. Takamata, G. W. Mack, and E. R. Nadel. "Measurement of plasma volume in rats with use of fluorescent-labeled albumin molecules." Journal of Applied Physiology 76, no. 1 (January 1, 1994): 485–89. http://dx.doi.org/10.1152/jappl.1994.76.1.485.
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We describe a method for measuring plasma volume (PV) in small animals that allows small sample sizes but does not require the use of radioisotopes and thus is a convenient approach for making repeated measurements. Texas Red covalently bound to albumin (TR-A) was used in a typical indicator-dilution technique to measure PV. The relative fluorescent intensity of TR-A is linear to its concentration (up to 0.15 mg/ml) at an excitation lambda of 590 nm and an emission lambda of 610 nm. Catheters were inserted through the right jugular vein of anesthetized rats and threaded into the vena cava. A 0.5-ml control blood sample was taken, a measured quantity of TR-A was injected, and the catheter was flushed with saline. A 0.5-ml postinjection sample was taken 5 min after TR-A injection. PV was calculated by comparing the difference between the relative fluorescent intensity of control and postinjection plasma samples to a standard. The PV of 22 rats [362 +/- 14 (SE) g] was 14.1 +/- 0.4 ml (39.6 +/- 0.9 ml/kg body wt) measured by the TR-A method and 12.8 +/- 0.4 ml (35.9 +/- 1.0 ml/kg body wt) measured by a standard radioiodinated albumin method. There was a strong correlation between PV measured by both methods in the same rat (r = 0.90, P < 0.01). Infusion experiments indicated that the TR-A method can detect acute changes in PV, and repeated measurements of PV made on a chronically instrumented rat demonstrated that the method can reliably measure PV on consecutive days.
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Xi, He, Yang Liu, Chun-Xue Yuan, Ye-Xin Li, Lei Wang, Xu-Tang Tao, Xiao-Hua Ma, Chun-Fu Zhang, and Yue Hao. "Through space charge-transfer emission in lambda (Λ)-shaped triarylboranes and the use in fluorescent sensing for fluoride and cyanide ions." RSC Advances 5, no. 57 (2015): 45668–78. http://dx.doi.org/10.1039/c5ra07912h.
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By introducing a twisted and non-conjugated Λ-shaped TB scaffold to triarylboranes, we provide an efficient strategy to develop a new class of organoboron compounds applied as colorimetric and ratiometric fluorescent sensors for fluoride and cyanide.
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Ioannou, P. C., and D. G. Konstantianos. "Fluorometric determination of magnesium in serum with 2-hydroxy-1-naphthaldehyde salicyloylhydrazone." Clinical Chemistry 35, no. 7 (July 1, 1989): 1492–96. http://dx.doi.org/10.1093/clinchem/35.7.1492.
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Abstract This simple, rapid, sensitive fluorometric method for determining magnesium is based on formation of a fluorescent complex of magnesium with 2-hydroxy-1-naphthaldehyde salicyloyl-hydrazone in alkaline ethanolic medium (lambda ex = 420 nm, lambda em = 460 nm). The detection limit of the method is 0.05 microgram/L (2 nmol/L) and the relative standard deviations (CVs) are 0.8%, 1.1%, and 2.5% at magnesium concentrations of 50, 5.0, and 0.50 microgram/L, respectively. The standard curve is linear from 0 to 250 micrograms/L (0-10 mumol/L). We investigated the chemistry of the reaction, and have applied the method to determine magnesium in 100-fold-diluted, otherwise untreated serum samples. Within-run CVs for the method were 1.7%, 1.1%, and 1.3% at mean magnesium concentrations of 15.7, 26.4, and 36.2 mg/L, respectively. Day-to-day CVs were 2.7%, and 3.6% at mean magnesium concentrations of 10.1 and 25.5 mg/L, respectively. Samples from 96 hospitalized patients in intensive-care units were analyzed by the proposed method (y) and by an automated colorimetric method involving Magon sulfonate reagent (x). Linear regression analysis of the results yielded: y = 1.01x - 0.04 (r = 0.982, Sxy = 0.90).
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Newmeyer, D. D., D. R. Finlay, and D. J. Forbes. "In vitro transport of a fluorescent nuclear protein and exclusion of non-nuclear proteins." Journal of Cell Biology 103, no. 6 (December 1, 1986): 2091–102. http://dx.doi.org/10.1083/jcb.103.6.2091.
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An in vitro system was developed that provides a quick microscopic assay for nuclear transport. The assay uses an extract of Xenopus eggs, normal or synthetic nuclei, and a fluorescently labeled nuclear protein, nucleoplasmin. This in vitro system accurately mimics in vivo nuclear transport, both in exclusivity and in the amount of accumulation observed (up to 17-fold). Selective accumulation of fluorescent nucleoplasmin is observed microscopically within 30 min with rat liver nuclei, Xenopus embryonic nuclei, regrown Xenopus sperm nuclei, or nuclei reconstituted in vitro from bacteriophage lambda DNA. This transport requires the signal domain of nucleoplasmin. Furthermore, the ability of nuclei to accumulate nucleoplasmin directly correlates with their ability to exclude the fluorescent non-nuclear proteins, FITC-immunoglobulin and phycoerythrin. An active transport model would predict that nuclear transport be temperature- and energy-dependent and that inhibition of transport by either low temperature or energy depletion would be reversible. Both predictions were confirmed in our system. Nucleoplasmin accumulation increases with temperature, while the protein is completely excluded at 0 degrees C. The effects of low temperature are reversible. As found for 125I-labeled nucleoplasmin (Newmeyer, D. D., J. M. Lucocq, T. R. Bürglin, and E. M. De Robertis, 1986, EMBO (Eur. Mol. Biol. Organ.) J., 5:501-510), transport of fluorescent nucleoplasmin is inhibited by ATP depletion. This effect is reversed by later ATP addition. Under ATP-depleted conditions non-nuclear proteins continue to be excluded. These results argue for a direct role of ATP in transport rather than for a simple role in preserving envelope integrity. In a first step towards defining the minimum requirements for a transport medium, egg extracts were depleted of membrane vesicles. Membrane-depleted extracts neither support transport nor maintain the integrity of the nuclear envelope.
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Bailey, G. G., and L. L. Needham. "Simultaneous quantification of erythrocyte zinc protoporphyrin and protoporphyrin IX by liquid chromatography." Clinical Chemistry 32, no. 12 (December 1, 1986): 2137–42. http://dx.doi.org/10.1093/clinchem/32.12.2137.
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Abstract A simple, rapid, specific, and sensitive isocratic "high-performance" liquid-chromatographic procedure is described for measuring protoporphyrin (PPIX) and zinc protoporphyrin (ZPP) in erythrocytes. A 30-microL whole-blood sample is treated with a solution of formic acid, deproteinized with acetone, and centrifuged. A 20-microL aliquot of the supernate is injected into a system consisting of a stationary phase of mu-Bondapak C18 and a mobile phase of acetone, methanol, water, and formic acid. ZPP and PPIX are detected fluorometrically (lambda ex = 417 nm, lambda em = 635 nm) within 6 min. The range of linearity extends beyond 10 mg/L for ZPP and 580 micrograms/L for PPIX. The detection limits for ZPP and PPIX are 11.9 micrograms/L (6.93 pg) and 2.55 micrograms/L (1.485 pg), respectively. The precision for ZPP and PPIX determinations averaged 2.86 and 5.59%, respectively, for within-day CVs and 4.98 and 8.14, respectively, for among-day CVs. Analytical recoveries averaged 97.2% for ZPP and 101.5% for PPIX. Interferences in the form of fluorescent quenching of ZPP and PPIX by hemin are avoided by chromatographic separation. We also used this method to determine the purity of commercially prepared ZPP, and compared the results obtained with this method with those from an extraction method.
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Yuan, Chun-Xue, Xu-Tang Tao, Lei Wang, Jia-Xiang Yang, and Min-Hua Jiang. "Fluorescent Turn-On Detection and Assay of Protein Based on Lambda (Λ)-Shaped Pyridinium Salts with Aggregation-Induced Emission Characteristics." Journal of Physical Chemistry C 113, no. 16 (March 26, 2009): 6809–14. http://dx.doi.org/10.1021/jp8111167.
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Guo, Lifang, Minggang Tian, Zhiyun Zhang, Qing Lu, Zhiqiang Liu, Guangle Niu, and Xiaoqiang Yu. "Simultaneous Two-Color Visualization of Lipid Droplets and Endoplasmic Reticulum and Their Interplay by Single Fluorescent Probes in Lambda Mode." Journal of the American Chemical Society 143, no. 8 (February 11, 2021): 3169–79. http://dx.doi.org/10.1021/jacs.0c12323.
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Elyamany, Ghaleb, Ali Matar Alzahrani, Eman Al Mussaed, Hassan Aljasem, Sultan Alotaibi, and Hatem Elghezal. "De NovoCD5 Negative Blastic Mantle Cell Lymphoma Presented with Massive Bone Marrow Necrosis without Adenopathy or Organomegaly." Case Reports in Hematology 2015 (2015): 1–6. http://dx.doi.org/10.1155/2015/146598.
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The recent World Health Organization (WHO) classification defines mantle cell lymphoma (MCL) as a distinct entity characterized by a unique immunophenotype and a molecular hallmark of chromosomal translocation t(11;14)(q13;q32). We report an unusual case of an advanced stage of CD5 negative MCL with a blastoid variant with a massive bone marrow (BM) necrosis as an initial presenting feature, with no adenopathy or hepatosplenomegaly. The pathologic features showed blastoid variant of MCL and flow cytometry showed that the tumor cells were CD5−, CD19+, CD20+, FMC-7+, CD23−, and lambda light chain restricted. Chromosomal analysis, using karyotype and fluorescent in situ hybridization (FISH), demonstrated karyotypic abnormalities in addition to the t(11;14). Our case study may be reported as a unique case of CD5− blastic MCL with unusual presentation and findings which made the diagnosis of MCL difficult.
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Weiss, Lukas, Christina Gruber, Ulrich Koller, Josefina Piñón-Hofbauer, Stefan Hainzl, Clemens Hufnagl, Thomas Melchardt, et al. "Spliceosome Mediated RNA Trans-Splicing for Targeting Kappa+ B-Cell Neoplasms." Blood 124, no. 21 (December 6, 2014): 3633. http://dx.doi.org/10.1182/blood.v124.21.3633.3633.
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Abstract Introduction B-cells express either kappa or lambda light chains but not both. B-cell directed therapies such as anti-CD20 antibodies deplete healthy and malignant CD20+ B-cells but spare CD20- plasma and myeloma cells. Furthermore anti-CD20 antibody mediated B-cell depletion can lead to severe hypoimmunoglobulinemia and thereby predispose for infections. Light-chain specific targeting would allow a more specific therapy of lymphomas and myeloma, with collateral damage limited to only half of healthy B-cells. Due to the heterogeneity of immunoglobulines, lymphoma specific therapy so far required customized solutions for every single patient. Despite having 40 variable and 5 joining regions, the kappa light chain has only 1 constant region (IGKC). Therefore the 5’ IGKC intron is conserved in kappa light chain pre-mRNA, regardless of any recombination or somatic hypermutation in the VJ-region. Spliceosome-mediated RNA trans-splicing allows endogenous pre-mRNA to be converted into a new gene product via exon replacement. This can be achieved by the introduction of an RNA trans-splicing molecule (RTMRTM) that binds to endogenous target RNA and induces trans-splicing between the target gene and the RTM. RTMs contain a binding domain (BD), which defines the target specificity, splicing elements for efficient trans-splicing, and the desired coding sequence. This proof-of-principle study was aimed at demonstrating the feasibility of light-chain specific targeting through spliceosome-mediated RNA trans-splicing. Methods This study was approved by the local ethics committee of the provincial government of Salzburg. Potential RTMs for trans-splicing were identified using a fluorescence screening procedure as previously described (Gruber C et al. Mol Cancer Ther. 2011 Feb;10(2):233-41.). Primary lymphoma and myeloma samples were collected during routine diagnostic testing. Total RNA isolation was performed with RNeasy® isolation kit (QIAGEN) and reverse transcription of RNA using the i-script cDNASynthesis Kit (Bio-Rad). Quantitative real-time polymerase chain reaction was performed using GoTaq® qPCR Master Mix (Promega). For western blotting, cell lysates were separated using 10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis. After transferring the proteins onto a nitrocellulose membrane, immune detection of specific proteins was performed using anti-Green Fluorescent Protein (GFP) antibodies and enhanced chemiluminescence detection. Results BD screening identified RTM 55 as the most efficient trans-splicing molecule and was therefore used for further experiments. Since the light chain locus undergoes substantial physiologic genetic changes in the process of VJ rearrangement and somatic hypermutation, we analysed whether these changes extend to the target sequence of the BD of RTM 55. Sequencing of the complete 5’ IGKC intron in primary kappa+ CLL (N=4), DLBCL (N=3) and myeloma cells (N=3) only showed occasional point mutations. In the following, effective endogenous trans-splicing was confirmed in 2 different kappa+ cell lines, the DLBCL cell line SUDHL4 and the CLL cell line MEC2. We could detect successful trans-splicing on the mRNA level and sequencing of PCR-products confirmed accurate trans-splicing with the last base of the J-region adjoining the first base of the GFP gene. Stable retroviral transfection of SUDHL4 cells also allowed the successful detection of the kappa light-chain-GFP fusion protein by western blotting. Surprisingly, the lambda+ myeloma cell line U266 as well as FACS-sorted healthy lambda+ B-cells showed high expression levels of kappa light chain mRNA. This leaky mRNA expression of the non-dominant light chain could not be seen in kappa+ cells, which may be explained by the programmed sequential activation of the kappa and lambda loci during B-cell development. Conclusions We could show that the kappa light chain can be specifically targeted by spliceosome-mediated RNA trans-splicing. The possibility to reprogram the light chain pre-mRNA offers numerous possible applications, such as suicide gene therapy for lymphoma and myeloma. The unexpectedly high expression of kappa mRNA in lambda+ cells but not vice-versa, raises the question whether the lambda light chain locus would offer an even better, since more specific target Disclosures No relevant conflicts of interest to declare.
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Zhang, Dianwei, Jiaqi Tang, and Huilin Liu. "Rapid determination of lambda-cyhalothrin using a fluorescent probe based on ionic-liquid-sensitized carbon dots coated with molecularly imprinted polymers." Analytical and Bioanalytical Chemistry 411, no. 20 (June 28, 2019): 5309–16. http://dx.doi.org/10.1007/s00216-019-01912-0.
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Inoue, Yukiko U., Yuki Morimoto, Mayumi Yamada, Ryosuke Kaneko, Kazumi Shimaoka, Shinji Oki, Mayuko Hotta, et al. "An Optimized Preparation Method for Long ssDNA Donors to Facilitate Quick Knock-In Mouse Generation." Cells 10, no. 5 (April 30, 2021): 1076. http://dx.doi.org/10.3390/cells10051076.
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Fluorescent reporter mouse lines and Cre/Flp recombinase driver lines play essential roles in investigating various molecular functions in vivo. Now that applications of the CRISPR/Cas9 genome-editing system to mouse fertilized eggs have drastically accelerated these knock-in mouse generations, the next need is to establish easier, quicker, and cheaper methods for knock-in donor preparation. Here, we reverify and optimize the phospho-PCR method to obtain highly pure long single-stranded DNAs (ssDNAs) suitable for knock-in mouse generation via genome editing. The sophisticated sequential use of two exonucleases, in which double-stranded DNAs (dsDNAs) amplified by a pair of 5’-phosphorylated primer and normal primer are digested by Lambda exonuclease to yield ssDNA and the following Exonuclease III treatment degrades the remaining dsDNAs, enables much easier long ssDNA productions without laborious gel extraction steps. By microinjecting these donor DNAs along with CRISPR/Cas9 components into mouse zygotes, we have effectively generated fluorescent reporter lines and recombinase drivers. To further broaden the applicability, we have prepared long ssDNA donors in higher concentrations and electroporated them into mouse eggs to successfully obtain knock-in embryos. This classical yet improved method, which is regaining attention on the progress of CRISPR/Cas9 development, shall be the first choice for long donor DNA preparation, and the resulting knock-in lines could accelerate life science research.
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Brodin, N. T., B. Jansson, G. Hedlund, and H. O. Sjögren. "Use of a monoclonal rat anti-mouse Ig light chain (RAMOL-1) antibody reduces background binding in immunohistochemical and fluorescent antibody analysis." Journal of Histochemistry & Cytochemistry 37, no. 7 (July 1989): 1013–24. http://dx.doi.org/10.1177/37.7.2499618.
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Rat monoclonal antibodies (MAb) directed to mouse Ig heavy and light chain determinants were produced. A rat anti-mouse light chain MAb (RAMOL-1) which bound to all (24/24) mouse Ig of the kappa light chain type and with varying strength to 4/4 lambda light chain-bearing Ig was evaluated as a general secondary reagent, together with two MAb that bound to the heavy chain of mouse IgG. They were conjugated with biotin or FITC and used in immunohistochemical and immunofluorescence assays to detect mouse monoclonal antibodies binding to antigens expressed in rat and human tissues and cells. As compared to commercially available polyclonal reagents, RAMOL-1 gave higher staining contrast by showing lower background staining and equal or higher staining of the primary MAb tested. This was a result of two main effects. First, crossreactivity with endogenous Ig and tissue type-specific determinants was eliminated. With polyclonal anti-mouse Ig reagents, binding to endogenous Ig was noted in vascular spaces and on Ig-bearing cells, and to rat gastric mucosa and epithelial tumor tissue in frozen tissue sections, even when diluted in high concentrations of serum homologous to the tissue. Second, binding of the secondary reagent was reduced to cells and tissues prone to have high nonspecific binding capability, such as monocytes/macrophages and formalin-fixed, paraffin-embedded tissue. Owing to unlimited and reproducible access to this homogeneous reagent, RAMOL-1 is used as second antibody to standardize the procedure used for immunohistochemical grading of human malignant tumors by determination of blood group antigen expression detected with mouse MAb.
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Semanaj, Valentina, Arbi Pecani, Teuta Dedej, Alma Barbullushi, Zamira Ylli, Teuta Curaj, Polikron Pulluqi, et al. "The Diagnostic Value of Flow Cytometry Imunophenotyping in an Albanian Patient Population with a Preliminary Clinical Diagnosis of Chronic Lymphocytic Leukemia." Open Access Macedonian Journal of Medical Sciences 2, no. 1 (March 15, 2014): 51–55. http://dx.doi.org/10.3889/oamjms.2014.009.
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Objective: Based on the flow cytometry multiparametric immunophenotyping methodology we studied some useful cell marker criteria needed for the practical differentiation of the chronic lymphocytic leukemia from other chronic limphoproliferative diseases with a leukemic component.Materials and Methods: The applied methodology is a four color flow cytometry multiparametric immunophenotyping technique using EDTA blood samples taken from 84 consecutive patients diagnosed with CLL through a preliminary clinical and white blood cell examination. The following fluorescent stained monoclonal antibodies were used: CD3, CD4, CD5, CD8, CD11c, CD19, CD20, CD23, CD25, FMC7 and kappa/lambda light chains.Results: From the 84 individuals tested, 2 out of them (2.4%) resulted with a abnormal T-cell population while 82 (97.6%) showed a pathological B cell line. 58 (69.1%) patients resulted with typical CLL markers (CD19+CD5+CD23+) while 5 (5.9%) of them presented a non typical chronic lymphocytic leukemia profile (CD19+CD5+CD23-). 19 (22.6%) out of patients displayed an abnormal CD19+CD5- B cell population. A statistically significant correlation was found between the clinical stage of CLL and the positivity for the CD38 marker (p=0.04).Conclusion: Flow cytometry immunophenotyping is a fundamental examination for the final diagnosis of chronic lymphocytic leukemia. The expression of CD38+ in CLL patients stands for a more advanced clinical stage.
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Lin, Y. C. James, and D. H. Evans. "Vaccinia Virus Particles Mix Inefficiently, and in a Way That Would Restrict Viral Recombination, in Coinfected Cells." Journal of Virology 84, no. 5 (December 23, 2009): 2432–43. http://dx.doi.org/10.1128/jvi.01998-09.
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ABSTRACT It is well established that poxviruses are subjected to genetic recombination, but attempts to map vaccinia virus genes using classical genetic crosses were historically confounded by high levels of experimental noise and a poor correlation between physical and genetic map distances. These virus-by-virus crosses also never produced the 50% recombinant progeny that should be seen in experiments involving distant markers. Poxviruses replicate in membrane-wrapped cytoplasmic structures called virosomes (or factories) and we have developed a method for tracking the development of these structures using live cell imaging and cells expressing phage lambda Cro protein fused to enhanced green fluorescent protein (EGFP). The EGFP-cro protein binds nonspecifically to DNA and permits live cell imaging of developing vaccinia virus factories. Using this method, we see virosomes first appearing about 4 to 5 h postinfection. The early virosomes exhibit a compact appearance and then, after a period of exponential growth lasting several hours, blur and start to dissipate in a process presumably linked to viral packaging. During the growth period, the virosomes migrate toward the nuclear periphery while colliding and fusing at a rate dependent upon the numbers of infecting particles. However, even at high multiplicities of infection (10 PFU/cell), we estimate ∼20% of the virosomes never fuse. We have also used fluorescence in situ hybridization (FISH) methods to study virosomes formed by the fusion of viruses carrying different gene markers. FISH showed that DNA mixes rather poorly within fused virosomes and the amount of mixing is inversely dependent on the time between virosome appearance and fusion. Our studies suggest that the intracellular movement and mixing of virosomes create constraints that reduce opportunities for forming recombinants and that these phenomena create outcomes reflected in classical poxvirus genetics.
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Mikala, Gabor, Emma Adam, Andras Kozma, Balazs Kapas, Sarolta Nahajevszky, and Tamas Masszi. "Striking Coincidence of IgD M-Protein and Translocation t(11;14) in Hungarian Myeloma Patients." Blood 108, no. 11 (November 16, 2006): 5046. http://dx.doi.org/10.1182/blood.v108.11.5046.5046.
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Abstract Multiple myeloma exhibiting production of IgD M-protein is a rare subset (appr. 1 %) of all patients. Translocation t(11;14) is, on the other hand, the most common primary translocation in myeloma that involves the IgH locus and cyclin D1, approximately 16–22% of all patients harbor this genetic rearrangement. This cytogenetic abnormality has been previously associated with nonsecretory behavior and also with production of IgE M-protein. Out of 317 myeloma patients treated in our department in 2004–2006, five patients had IgD M-protein (four belonged to IgD-lambda and one to IgD kappa subtype). Cytogenetic analysis by interphase fluorescent in situ hybridization (FISH) revealed the presence of the IgH/Cyclin D1 abnormality in all five of them (100%), while the prevalence of this translocation was 17% in the entire myeloma population seen by our ward and tested by FISH (132 patients). One IgD patient had a complex IgH/MYC/CyclinD1 translocation, also confirmed by conventional cytogenetic analysis of metaphases, while two of five patients had additional copies of IgH and Cyclin D1 genes, possibly indicating atypical translocations and/or hyperdiploidy. Although the number of patients with this rare disease in our report is low (5), we find the uniform presence of translocation t(11;14) a striking occurrence that may explain some of the unusual biological properties of this myeloma subtype.
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Turnacioglu, K. K., B. Mittal, G. A. Dabiri, J. M. Sanger, and J. W. Sanger. "An N-terminal fragment of titin coupled to green fluorescent protein localizes to the Z-bands in living muscle cells: overexpression leads to myofibril disassembly." Molecular Biology of the Cell 8, no. 4 (April 1997): 705–17. http://dx.doi.org/10.1091/mbc.8.4.705.
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Cultures of nonmuscle cells, skeletal myotubes, and cardiomyocytes were transfected with a fusion construct (Z1.1GFP) consisting of a 1.1-kb cDNA (Z1.1) fragment from the Z-band region of titin linked to the cDNA for green fluorescent protein (GFP). The Z1.1 cDNA encodes only 362 amino acids of the approximately 2000 amino acids that make up the Z-band region of titin; nevertheless, the Z1.1GFP fusion protein targets the alpha-actinin-rich Z-bands of contracting myofibrils in vivo. This fluorescent fusion protein also localizes in the nascent and premyofibrils at the edges of spreading cardiomyocytes. Similarly, in transfected nonmuscle cells, the Z1.1GFP fusion protein localizes to the alpha-actinin-containing dense bodies of the stress fibers in vivo. A dominant negative phenotype was also observed in living cells expressing high levels of this Z1.1GFP fusion protein, with myofibril disassembly occurring as titin-GFP fragments accumulated. These data indicate that the Z-band region of titin plays an important role in maintaining and organizing the structure of the myofibril. The Z1.1 cDNA was derived from a chicken cardiac lambda gt11 expression library, screened with a zeugmatin antibody. Recent work has suggested that zeugmatin is actually part of the N-terminal region of the 81-kb titin cDNA. A reverse transcriptase polymerase chain reaction using a primer from the distal end (5' end) of the Z1.1 zeugmatin cDNA and a primer from the nearest known proximal (3' end) chicken titin (also called connectin) cDNA resulted in a predicted 0.3-kb polymerase chain reaction product linking the two known chicken titin cDNAs to each other. The linking region had a 79% identity at the amino acid level to human cardiac titin. This result and a Southern blot analysis of chicken genomic DNA hybridized with Z1.1 add further support to our original suggestion that zeugmatin is a proteolytic fragment from the N-terminal region of titin.
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Weiss, David S., Joseph C. Chen, Jean-Marc Ghigo, Dana Boyd, and Jon Beckwith. "Localization of FtsI (PBP3) to the Septal Ring Requires Its Membrane Anchor, the Z Ring, FtsA, FtsQ, and FtsL." Journal of Bacteriology 181, no. 2 (January 15, 1999): 508–20. http://dx.doi.org/10.1128/jb.181.2.508-520.1999.
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ABSTRACT Assembly of the division septum in bacteria is mediated by several proteins that localize to the division site. One of these, FtsI (also called penicillin-binding protein 3) of Escherichia coli, consists of a short cytoplasmic domain, a single membrane-spanning segment, and a large periplasmic domain that encodes a transpeptidase activity involved in synthesis of septal peptidoglycan. We have constructed a merodiploid strain with a wild-type copy offtsI at the normal chromosomal locus and a genetic fusion of ftsI to the green fluorescent protein (gfp) at the lambda attachment site. gfp-ftsI was expressed at physiologically appropriate levels under control of a regulatable promoter. Consistent with previous results based on immunofluorescence microscopy GFP-FtsI localized to the division site during the later stages of cell growth and throughout septation. Localization of GFP-FtsI to the cell pole(s) was not observed unless the protein was overproduced about 10-fold. Membrane anchor alterations shown previously to impair division but not membrane insertion or transpeptidase activity were found to interfere with localization of GFP-FtsI to the division site. In contrast, GFP-FtsI localized well in the presence of β-lactam antibiotics that inhibit the transpeptidase activity of FtsI. Septal localization depended upon every other division protein tested (FtsZ, FtsA, FtsQ, and FtsL). We conclude that FtsI is a late recruit to the division site, and that its localization depends on an intact membrane anchor.
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Carlile, A. J., L. V. Bindschedler, A. M. Bailey, P. Bowyer, J. M. Clarkson, and R. M. Cooper. "Characterization of SNP1, a Cell Wall-Degrading Trypsin, Produced During Infection by Stagonospora nodorum." Molecular Plant-Microbe Interactions® 13, no. 5 (May 2000): 538–50. http://dx.doi.org/10.1094/mpmi.2000.13.5.538.
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Stagonospora (= Septoria) nodorum when grown in liquid culture with wheat cell walls as the sole carbon and nitrogen source secretes numerous extracellular depoly-merases, including a rapidly produced, alkaline, trypsin-like protease (SNP1). The enzyme was purified 417-fold by cation exchange chromatography and has a molecular mass of 25 kDa on sodium dodecyl sulfate gels, pI 8.7, and pH optimum of 8.5. It cleaved peptide bonds on the carboxyl side of lysine or arginine, was strongly inhibited by the trypsin inhibitors aprotinin and leupeptin and weakly by phenylmethylsulfonyl fluoride, and its activity was stimulated by calcium. SNP1 has the characteristic, conserved, fungal, trypsin N terminus. Polymerase chain reaction (PCR) primers based on this sequence and the conserved trypsin active site were used to amplify a DNA fragment that facilitated isolation of the corresponding genomic clone from a lambda library of S. nodorum. The full-length sequence confirmed its identity as a trypsin-like protease containing the N-terminal sequence of the previously purified enzyme. Infected leaf tissue contained a protease, not present in controls, that coeluted with the fungal trypsin from cation exchange, and had properties (pI and inhibitor characteristics) similar to those of the fungal trypsin. SNP1 expression in planta was detected by Northern (RNA) blotting, reverse transcription PCR, and green fluorescent protein confocal microscopy. SNP1 released hydroxyproline from wheat cell walls. The release of hydroxyproline, together with its early expression in planta, suggests that SNP1 participates in the degradation of host cell walls during infection.
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Bryce, A. H., R. P. Ketterling, M. A. Gertz, R. A. Kyle, J. A. Lust, R. Fonseca, M. Lacy, et al. "Cytogenetic analysis using multiple myeloma targets in POEMS syndrome." Journal of Clinical Oncology 25, no. 18_suppl (June 20, 2007): 8116. http://dx.doi.org/10.1200/jco.2007.25.18_suppl.8116.
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8116 Background: POEMS syndrome, or osteosclerotic myeloma, is an uncommon plasma cell disorder associated with Peripheral neuropathy, Organomegaly, Endocrinopathy, M protein, and Skin changes, as well as bone lesions, Castlemans disease, and edema. The pathogenesis of POEMS is poorly understood, and its clinical course is distinct from Multiple Myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS). While various cytogenetic abnormalities have been well described in MM and MGUS, we provide the first report of testing for these abnormalities in POEMS syndrome. Methods: Using a prospectively held dysproteinemia database, we reviewed all cases of POEMS seen at our institution, identifying 36 with metaphase cytogenetic testing results, and 9 with fluorescence in situ hybridization (FISH) testing results. Combined cytoplasmic immunoglobulin/FISH testing (cIg- FISH) was performed with fluorescent-tagged antibodies to kappa/lambda immunoglobulin and FISH probes corresponding to the following anomalies: trisomies 3, 7, 9, 11, 15 and 17; del13/del13q; 14q32 split/del14; and t(11;14). Results: Five patients had insufficient plasma cells for cIg-FISH, and 9 were adequate for testing. Monosomy 13 was seen in 5 of the 9 cIg-FISH samples of which 2 of these cases also had apparent monosomy 14. cIg-FISH in one patient revealed trisomy 3 and 7. No abnormalities were seen at 11q13 (CCND1), 17p13 (p53) or translocations involving 14q32 (IGH). Thrity-eight total karyotypes analyses were attempted, though two showed an insufficient number of suitable metaphases. Of the 36 karyotype analyses, 31 were normal, 2 displayed characteristic plasma cell anomalies (hyperdiploid with structural anomalies), 2 treated patients displayed del20q anomalies and 1 patient had a constitutional 13;14 translocation. Conclusion: We are the first to report del13 as a possibly common abnormality in POEMS syndrome, detected by cIg-FISH in 5 of 9 (55%) samples with sufficient plasma cells, similar to the rate observed in MM and MGUS. The lack of abnormalities at 14q32 (IgH locus) is in contrast to MM and MGUS. Abnormal karyotypes, consistent with dividing plasma cells, are rare in POEMS, observed in only 2 of 36 (5.5%) cases. These observations could possibly lend insight into all three disorders. No significant financial relationships to disclose.
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Andrade, L. E., E. K. Chan, I. Raska, C. L. Peebles, G. Roos, and E. M. Tan. "Human autoantibody to a novel protein of the nuclear coiled body: immunological characterization and cDNA cloning of p80-coilin." Journal of Experimental Medicine 173, no. 6 (June 1, 1991): 1407–19. http://dx.doi.org/10.1084/jem.173.6.1407.
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Antibodies producing an unusual immunofluorescent pattern were identified in the sera of patients with diverse autoimmune features. This pattern was characterized by the presence of up to six round discrete nuclear bodies in interphase cell nuclei. Immunoblotting analysis showed that these sera recognized an 80-kD nuclear protein, and affinity-purified anti-p80 antibody from the protein band reproduced the fluorescent staining of nuclear bodies. Colloidal gold immunoelectron microscopy showed that the affinity-purified anti-p80 antibody recognized the coiled body, an ultramicroscopic nuclear structure probably first described by the Spanish cytologist Ramon y Cajal. Five cDNA clones were isolated from a MOLT-4 cell lambda gt-11 expression library using human antibody and oligonucleotide probes. The longest cDNA insert was 2.1 kb and had an open reading frame of 405 amino acids. A clone encoding a 14-kD COOH-terminal region of the protein was used for expression of a beta-galactosidase fusion protein. An epitope was present in this COOH-terminal 14-kD region, which was recognized by 18 of 20 sera with anti-p80 reactivity, and affinity-purified antibody from the recombinant protein also reacted in immunofluorescence to show specific staining of the coiled body. This is the first demonstration and molecular cloning of a protein that appears to have particular identification with the coiled body, and it was designated p80-coilin. Autoantibody to p80-coilin may be useful for the elucidation of the structure and function of the coiled body, and the availability of a cDNA sequence could be helpful in further studies to clarify the clinical significance of this autoantibody response.
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Rajput, Sheerien Kareem, and El-Nasir M. A. Lalani. "Establishment and Characterization of a Myeloma Cell Line- Aku-MY01." Blood 134, Supplement_1 (November 13, 2019): 5535. http://dx.doi.org/10.1182/blood-2019-132238.
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Introduction: Multiple myeloma (MM) is characterized by expansion of neoplastic plasma cells (PCs). Disease presentation is heterogenous with hypercalcemia, renal failure, osteolytic lesions or a combination thereof. The overall survival of patients and their response to treatment has improved considerably however, MM stays incurable. Patients develop treatment resistance and progress to relapse. Disease progression to treatment resistance and relapse are attributed to complex signaling molecules. In vitro experiments on cell lines are pivotal to determine the functional characterization of these molecules leading to identification of potential therapeutic targets. Cell lines have served as instruments for research in varied disciplines including cancer. Currently, majority of the cell lines in cancer research are from European origin and to a lesser extent from Africans, Asians and Hispanic populations. This lacuna of inadequate racial and genetic representation leads to major consequences such as delays in the benefits of precision medicine, drug efficacy and partial understanding of molecular aspects of the disease. The study was undertaken to establish and characterize a cell line from a patient diagnosed with MM. Methods AKU-MY01 was established from a 38-year male patient diagnosed with ISS stage III, kappa light chain myeloma in 2013. Plasma cells were isolated from the BM aspirate using sterile conditions and cultured in RPMI-1640 supplemented with 10% FBS. Conditioned media from urinary bladder carcinoma cell line 5637 was used to provide growth factors. Isolated suspension cells were observed in culture which were subcultured by splitting in a 1:3 ratios. Detailed characterization of AKU-MY01 was undertaken using gene expression analysis, population doubling time (PDT), determination of clonality, fluorescent in situ hybridization, immunocytochemistry (ICC), karyotyping, transwell assay and short tandem repeat (STR) analysis. Results: AKU-MY01 showed expression of ABCG2dim, Oct4, CD38, CD138, CD19, CD20, CD45, CD56 dim, XBP1, MUC1, EBNA1, and monoclonal Lambda light chain. AKU-MY01 exhibited a human diploid karyotype with 46XY and IGH deletions/translocation in 20% of the cells. It has a PDT of 48-53 hrs. and high invasive potential (15.6%) compared to other MM cell lines. Expression of EBNA at mRNA level was further confirmed through fluorescent ICC. The results from Immunofluorescent ICC showed clusters of tumor cells with concordant expression of membranous CD138 and cytosolic and nuclear expression of LMP1. Further, the expression of LMP1 in CD138 positive tumor cells was confirmed in Patient's trephine from whome AKU-MY01 was derived. Conclusion: To the best of our knowledge, AKU-MY01 is the first MM cell line characterized for LMP1 expression in CD138 expressing myeloma cells. Salient features of AKU-MY01 which make it a unique addition to the existing repertoire of MM cell lines are a) Asian origin, b) diploid karyotype and c) expression of EBV protein and mRNA. Further studies using this cell line model may contribute to understand the biology of EBV positive MM cases. Disclosures No relevant conflicts of interest to declare.
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Tsunekawa, Norikazu, Ichiro Hanamura, Hidesuke Yamamoto, Hisao Nagoshi, Motohiro Wakabayashi, Natsumi Sakamoto, Tomohiro Horio, et al. "Establishment and Characterization of a Novel Human Cell Line of Triple Hit Lymphoma, AMU-ML1, Carrying t(2;18)(p11.2;q21) and t(3;8;14)(q27;q24;q32) That Involve BCL2, BCL6 and MYC." Blood 118, no. 21 (November 18, 2011): 5210. http://dx.doi.org/10.1182/blood.v118.21.5210.5210.
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Abstract Abstract 5210 Mature B-cell lymphomas with both BCL2 and MYC translocations to IG loci are rarely identified and most of them are classified as B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma (IL) in the new World Health Organization classification. In this study, we established a novel human lymphoma cell line, AMU-ML1, from pericardial effusion (PE) of a patient with IL before the initiation of chemotherapy and analyzed its characters. A 60-year-old male was admitted to our hospital because of PE and diagnosed as having IL from the atypical lymphocytes in PE. He was treated with rituximab plus hyper-CVAD and other regimens but died of lymphoma approximately 10 months after diagnosis without reaching complete remission. The cells from patient's PE at diagnosis were cultured in RPMI 1640 supplemented with 20% heat-inactivated fetal bovine serum (FBS). After 5 months of culture, cell proliferation became continuous with 10% FBS and the cell line was designated as AMU-ML1 (Aichi Medical University, malignant lymphoma, no. 1) after confirmation that the cells began growing again after the conventional freeze-thaw procedure. AMU-ML1 cells were positive for CD10, CD19, CD20, CD79a, HLA-DR and cytoplasmic lambda chain and negative for CD3, CD4, CD5, CD8, CD13, CD23, CD33 and CD56 by flow cytometry analysis and showed a complex karyotypes including t(2;18)(p11.2;q21) and t(3;8;14)(q27;q24;q32) by G-banding analysis. This profile is consistent with the profile of the patient's cells. Spectral karyotyping and fluorescent in situ hybridization analysis of AMU-ML1 cells revealed that t(2;18)(p11.2;q21) was IGL/BCL2 and t(3;8;14)(q27;q24;q32) was BCL6/MYC, MYC/IGH and IGH/BCL6. Subcutaneous transplantation of AMU-ML1 cells into NOD/scid mice treated with anti-asialo GM1 antibody resulted in formation of primary tumors. Thus, the AMU-ML1 cell line is useful for studying the biological consequences of IL with triple hit of BCL2, BCL6 and MYC, and possibly invasion to PE of lymphoma. Disclosures: No relevant conflicts of interest to declare.
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Zhang, Jing, Mangjuan Tao, and Yan Jin. "An enzyme-aided amplification strategy for sensitive detection of DNA utilizing graphene oxide (GO) as a fluorescence quencher." Analyst 139, no. 13 (2014): 3455–59. http://dx.doi.org/10.1039/c4an00151f.
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A facile, sensitive and rapid method has been developed for detection of disease-related DNA based on lambda exonuclease-aided signal amplification by utilizing graphene oxide (GO) as a fluorescence quencher.
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Gebhard, D. F., A. Mittelman, C. Cirrincione, H. T. Thaler, and B. Koziner. "Comparative analysis of surface membrane immunoglobulin determination by flow cytometry and fluorescence microscopy." Journal of Histochemistry & Cytochemistry 34, no. 4 (April 1986): 475–81. http://dx.doi.org/10.1177/34.4.3081624.
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The analysis of membrane surface immunoglobulin (SmIg) on B lymphocytes was carried out in 59 normal individuals and nine patients with B-cell non-Hodgkin's lymphomas by conventional immunofluorescence microscopy and flow cytometry. Five channel settings of a cytofluorograph were evaluated (100, 150, 200, 250, 300) and the mean and standard deviation of the percent positive cells were calculated and compared to the mean and standard deviation of the microscope reading. On the basis of the relative fluorescence reactivity, we were able to determine a fluorescence intensity at which the results of flow cytometry and fluorescence microscopy were comparable. In normal individuals, for cells expressing surface Ia, the channel giving similar results to that of fluorescence microscopy was 150; for kappa and lambda chains, channel 200; for Fab'PV, channel 200; and for IgM, channel 250. In patients with B-cell non-Hodgkin's lymphomas, for cells expressing surface Ia the channel giving similar results to that of fluorescence microscopy was 100; for kappa, channel 100; for lambda, channel 200; for Fab'FV, channel 150; and for IgM, channel 150. Flow cytometric analysis of SmIg appears to be superior to fluorescence microscopy in efficiency, and has the added advantages of being a rapid, sensitive, and objectively quantitative methodology.
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Jackman, Rachael P., Douglas Bolgiano, Mila Lebedeva, Sherrill J. Slichter, and Philip J. Norris. "C1q-Binding HLA Antibodies Do Not Predict Platelet Transfusion Failure in TRAP Study Participant." Blood 124, no. 21 (December 6, 2014): 1562. http://dx.doi.org/10.1182/blood.v124.21.1562.1562.
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Abstract Introduction: In the Trial to Reduce Alloimmunization to Platelets (TRAP) study, 101 of the 530 subjects became clinically refractory (CR) to platelet transfusions in the absence of detectable antibodies against HLA as measured by the lymphocytotoxicity assay (LCA). Using more sensitive bead-based detection methods we have previously demonstrated that while many of these LCA- patients do have anti-HLA antibodies, that these low to moderate level antibodies do not predict refractoriness. In addition to being less sensitive then bead based methods, the LCA screen only detects complement-binding antibodies. As these antibodies could be important for platelet rejection, we assessed if previously undetected complement-binding antibodies could account for some of the refractoriness seen in LCA- patients. Methods: 169 LCA- (69 CR, 100 non-CR) and 20 LCA+ (10 CR, 10 non-CR) subjects were selected from the TRAP study. Anti-class I HLA IgG and C1q binding antibodies were measured in serum or plasma using two different multi-analyte, semi-quantitative, bead-based fluorescent antibody detection assays: the LabScreen mixed Luminex assay, and the LabScreen single antigen class I assay with or without added EDTA (One Lambda). Groups were compared using an unpaired t-test, a=0.05, and correlation between variables was also assessed. Results: New measurements of anti-class I HLA IgG antibodies reliably reproduced earlier data with a strong correlation between the old and new measurements (R2=0.9736, p<0.0001). While some of the LCA- subjects did have detectable C1q-binding anti-class I HLA antibodies, and some LCA+ subjects did not, levels of these antibodies were significantly higher among LCA+ subjects (p<0.0001). Levels of C1q-binding anti-class I HLA antibodies were not significantly different between CR and non-CR among either the LCA- or LCA+ subjects. Conclusions: While complement-binding anti-class I HLA antibody levels were higher in the LCA+ subjects, higher levels of these antibodies were not seen in CR LCA+ patients as compared with non-CR LCA+ patients. Complement-binding anti-class I HLA antibodies do not account for refractoriness seen among the LCA- TRAP subjects. This work confirms that low to mid level anti-class I antibodies do not drive refractoriness to platelets, and suggests that antibody-independent mechanisms cause refractoriness in patients lacking higher levels of anti-HLA antibodies. Disclosures No relevant conflicts of interest to declare.
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Fasurová, N., and L. Pospíšilová. "Spectroscopic characteristics of humates isolated from different soils." Soil and Water Research 6, No. 3 (September 19, 2011): 147–52. http://dx.doi.org/10.17221/21/2010-swr.
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Spectral characterisation of soil humic substances is one of the important methods for their quality identification. In this work, two optical methods (UV-VIS and SFS) were used. The absorbance in the spectral range of 300–700 nm was measured using spectrometer Varian Cary 50 Probe. Fluorescence (SFS) in the range of 255–655 nm was performed by spectrofluorimeter Aminco Bowman. Five Czech soil humates samples (Leptic Cambisol, Haplic Cambisol, Eutric Cambisol 1-arable soil, Eutric Cambisol 2-grassland, Haplic Chernozem) were compared. The basic soil properties were determined by the commonly used methods. Colour indexes (Q<sub>4/6</sub>) were calculated from the absorbance of humic substances in UV- VIS spectral range. Fluorescence indexes (F) were calculated from SFS spectra at Δλ = 55 nm (as a ratio RFI<sub>468</sub>/RFI<sub>522</sub>). Also, the classical method of humic substances fractionation to assess their quality was applied. The comparison is given of the calculated parameters from different spectral regions and humic substances fractionation. The results showed linear correlation between carbon content (C weigth %) and fluorescence indexes (R<sup>2 </sup>= 0.91), between total organic carbon content in soil and fluorescence indexes (R<sup>2 </sup>= 0.92), as well as between colour indexes (Q<sub>4/6</sub>) and humic substances content. The same main fluorophores at the wavelengths λ<sub>ex</sub>/λ<sub>em</sub>= 467/522 nm were observed in all soil humates samples.
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Bryce, Alan H., Rhett Ketterling, Morie Gertz, Steve Zeldenrust, Martha Lacy, Shaji Kumar, Suzanne Hayman, et al. "14q32 Abnormalities and 13q Deletions Are Common in Primary Systemic Amyloidosis Using Cytoplasmic Immunoglobulin Fluorescence In Situ Hybridization (cIg-FISH)." Blood 110, no. 11 (November 16, 2007): 2477. http://dx.doi.org/10.1182/blood.v110.11.2477.2477.
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Abstract Background: Primary systemic amyloidosis is an uncommon plasma cell dyscrasia characterized by organ deposition of immunoglobulin light or heavy chain fragments. It is related to the other plasma cell dyscrasias such as multiple myeloma and monoclonal gammopathy of undetermined significance. cIg-FISH on bone marrow plasma cells has become well established in the initial evaluation of myeloma as the t(4;14), t(14;16),13q-, and the 17p- abnormality are of major prognostic value. The corresponding data does not exist in amyloidosis, as there are only three small studies published to date. Methods: Using a prospectively held dysproteinemia database, we reviewed all cases of amyloidosis seen at our institution who had cIg-FISH or cytogenetics performed as part of their routine clinical testing. Three hundred thirty nine patients were identified with amyloidosis and cytogenetic testing. Of these 69 had cIg-FISH testing results. cIg-FISH was performed with fluorescent-tagged antibodies to kappa/lambda immunoglobulin and FISH probes corresponding to the following anomalies: trisomies 3, 7, 9 and 15; del17p/-17; -13/del13q; 14q32 split; and t(11;14). CCND1/IGH. If an IGH rearrangement was detected that did not involve CCNDI, we also evaluated t(4;14) or IGH/FGFR3 and t(14;16) or IGH/c-MAF. These probes constitute the standard screening panel used in MM at our institution. Results: The study population had a median age of 61 years with a male to female ratio of 1.6:1. Median follow up time was 13.5 months. By conventional cytogenetics, 88% of patients had no detectable abnormality. In contrast, only 33% were normal by cIg-FISH. The abnormalities demonstrated were 28% 13-/del13q, 42% IgH translocation, 33% t(11;14), and 3% t(14;16). No patients had a -17/del 17p- or t(4;14). Nine patients had del13q/-13 with an IgH translocation, with 7 of these having t(11;14). Both t(14;16) patients had del 13q. A trend towards increased mortality was seen in patient with any abnormality (p=0.11), but this could not be demonstrated for any single abnormality, likely due to lack of statistical power. There was no correlation between renal involvement or cardiac involvement and cIg-FISH results. Conclusion: The majority of chromosomal abnormalities in amyloidosis are undetected by conventional cytogenetics. With further study, chromosomal abnormalities may carry the same prognostic value in amyloidosis that they currently enjoy in myeloma. This effect may be independent of currently recognized risk factors. Chromosomal abnormalities in Amyloidosis as detected by cIg-FISH Abnormality Amyloidosis % (n=69) Normal 33(23) −13/del13q 28(19) IgH Abnormality 42(29) t(4;14) 0(0) t(11;14) 33(23) t(14;16) 3(2) del 17 0(0) Other 3(2)
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Lamont, Elizabeth B., Andrew J. Yee, Stuart L. Goldberg, and Andrew D. Norden. "Chromosome 1q Amplification Is Associated with a History of Prior Malignancies Among Patients Newly Diagnosed with Multiple Myeloma." Blood 134, Supplement_1 (November 13, 2019): 2193. http://dx.doi.org/10.1182/blood-2019-125407.
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Background: Over the past 20 years, observational data from usual care clinical oncology settings has been leveraged to inform estimates of cancer treatment-associated benefits and risks among patients not treated on clinical trials. Increasing genomic testing to inform treatment decisions in usual care settings now meaningfully augments traditional observational data, positioning it to provide insights beyond clinical care into tumor biology. We studied patients with newly diagnosed multiple myeloma (MM), comparing cytogenetic test patterns according to history of prior malignancy. Methods: In this retrospective cohort study, we identified 2,380 patients from the COTA real-world database (RWD) who were newly diagnosed with MM in the years 2010-2018. The COTA RWD is a de-identified composite of both abstracted electronic health record and administrative data pertaining to patients receiving their cancer care at one of COTA's clinical oncology practice partners. Among these patients, 1769 (74%) had evidence of MM-associated cytogenetic testing with fluorescent in-situ hybridization (FISH) within the 120 days surrounding their date of diagnosis. The 1,769 patients form the analytic cohort. We compared patients' FISH results for t(4;14), deletion(17p), t(14;16), deletion(13), t(14;20), t(6;14), t(11;14), deletion (1p), and amplification(1q) according to their history of prior malignancy. Results: Within the cohort, 263 prior malignancies were identified in 241 patients (14%, 241/1,769). Two-hundred and twenty-one patients (92%) had one prior malignancy, 28 (7.9%) had two prior malignancies, and one (<1%) had four prior malignancies. The most common prior malignancies were prostate (n=50), breast (n=19), melanoma (n=14), skin (n=13), and cervix (n=6). Amplification of the long arm of chromosome one (amp(1q)) was noted in 31% of patients (75/241) with a prior malignancy vs. 24% of patients (370/1,528) without (chi2 test p=0.02). Overall 25% of patients had amp(1q). No other translocations, amplifications, deletions were associated with prior cancers. A non-parametric test for trend revealed a strong positive association between patients' malignancy count (range 0-4) and amp1q (p<0.01). MM patients with prior lymphomas and prior melanomas also had high rates of amp(1q), though these were not significantly different from patients without these prior malignancies. In a multivariable logistic regression model that adjusted for patient demographic attributes, other known potentially collinear MM poor prognostic factors (i.e., revised ISS stage, IgA sub-type, lambda light chains) and adjusted standard errors for clustering of patients within treatment settings, a history of prostate cancer remained clinically and statistically significantly positively associated with amp(1q) (OR 2.1, 95% CI: 1.9-2.2) as did history of two or more prior malignancies (OR 2.8, 95% CI: 2.3-3.3). Of note, amp(1q) was positively associated with IgA subtype (OR 1.5, 95% CI: 1.3-1.6) and the presence of lambda subtype (OR 1.3, 95%CI: 1.3-1.4). Conclusions: Using RWD, we found that newly diagnosed MM patients with histories of prostate cancer and those with two or more prior malignancies were more likely to have amp(1q), a poor prognostic marker in MM. Gains in 1q have previously been identified among patients with prostate and lymphoid cancers, but to our knowledge this is the first study to identify an association with a prior history of cancer, especially prostate cancer, and amp(1q) in MM. This relationship is worth further exploration of whether there is a common pathway associated with for example risk of prostate cancer and amp(1q) in MM. Clinical trials are less likely to answer this question as patients with prior malignancies are often excluded from enrollment. Overall, the results reported suggest that RWD is an efficient and comparatively inexpensive tool to support research in cancer biology through hypothesis generating and testing analyses of linked real-world phenotypic and genotypic data. Disclosures Lamont: COTA: Employment. Yee:Celgene: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; Takeda: Consultancy; Adaptive: Consultancy; Amgen: Consultancy, Honoraria. Goldberg:Cancer Outcomes Tracking and Analysis (COTA) Inc.: Equity Ownership; COTA: Equity Ownership; Bristol-Myers Squibb: Consultancy. Norden:COTA: Employment, Equity Ownership.
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Shimomura, O., and A. Shimomura. "Halistaurin, phialidin and modified forms of aequorin as Ca2+ indicator in biological systems." Biochemical Journal 228, no. 3 (June 15, 1985): 745–49. http://dx.doi.org/10.1042/bj2280745.
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Two kinds of aequorin-type photoproteins, i.e., halistaurin and phialidin, and four kinds of modified forms of aequorin, i.e., products of acetylation, ethoxycarbonylation, fluorescamine-modification and fluorescein labelling, were prepared. The modified forms of aequorin were more sensitive to Ca2+ than was aequorin in their Ca2+-triggered luminescence reactions, whereas halistaurin and phialidin were less sensitive. The emission maxima of luminescence were all within a wavelength range 450-464 nm, except for fluorescein-labelled aequorin, which emitted yellowish light (lambda max. 520 nm). A new technique of measuring Ca2+ concentration is suggested.
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Burlacu, S., P. A. Janmey, and J. Borejdo. "Distribution of actin filament lengths measured by fluorescence microscopy." American Journal of Physiology-Cell Physiology 262, no. 3 (March 1, 1992): C569—C577. http://dx.doi.org/10.1152/ajpcell.1992.262.3.c569.
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We analyzed the distribution of actin filament lengths by optical microscopy (OM). OM avoids possible alterations in the size or structure of actin filaments occurring during sample preparation for electron microscopy (EM). Images of F-actin labeled with tetramethylrhodamine isothiocyanate (TRITC)-phalloidin were analyzed for both size distribution and flexibility. In the standard buffer [25 mM potassium acetate, 4 mM MgSO4, 25 mM tris(hydroxymethyl)aminomethane acetate, pH 7.5, 20 mM beta-mercaptoethanol] filaments did not aggregate into bundles and remained stable at nanomolar concentrations for at least 1 h. At the same concentration, actin labeled directly with rhodamine (no phalloidin) formed unstable filaments whose average length decreased with time. The number average length of TRITC-phalloidin labeled filaments (Ln) was 4.90 microns, the ratio (rho) of the weight average length to the number average length was 2.06, and the correlation length (1/lambda) was 8.33 microns. These parameters were in good agreement with the values determined by EM for filaments shorter than 8 microns. Passing G-actin through a Sephadex G-150 column before polymerization did not have a significant effect on the distribution of lengths but made filaments more stiff (1/lambda = 12.5 microns). Millimolar concentration of ATP increased the correlation length, and gelsolin had the expected fragmenting effect on filaments. These results show that OM can be used as a fast and reliable method to analyze the distribution and flexibility of actin filaments and suggest that, in spite of extensive manipulation of actin filaments during sample preparation, EM is a valid tool for determination of size parameters of actin filaments.
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Sun, Xuemei, Junhao Chen, Jingyan Xu, Yifen Zhang, and Wei Xie. "Multiple Parameter Flow Cytometric Immunophenotyping Is Helpful in the Diagnosis of Patients with Chronic Lymphoproliferative Diseases." Blood 108, no. 11 (November 16, 2006): 4632. http://dx.doi.org/10.1182/blood.v108.11.4632.4632.
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Abstract The new WHO classification of lymphoid malignancies can become a big challenge to pathologists, owing to technical limitations of conventional pathologic diagnosis and immunohistochemistry. Multiple parameter analysis by flow cytometry (FCM) could provide a potent tool in improving the diagnostic status of chronic lymphoproliferative diseases(CLPDs). To evaluate its role in improving the accuracy and efficacy of diagnosis, we studied the immunophenotype by FCM in a total of 214 samples, including lymph nodes, peripheral blood, bone marrow and cerebrospinal fluid samples, in 186 patients with suspected mature B or T cell neoplasmas. A total of 102 male and 84 female patients were included in this study. The medium age was 49 years old, ranging from 3 to 82. Samples were prepared in routine fashion for morphology, pathology and immunohistochemistry, as LCA, L26, UCHL-1, CD5, Cyclin D1, CD10, Bcl-2, CD20, CD3, TdT and CD79α stains were performed by traditional APAAP method. A diagnosis was made according to the new WHO classification for lymphoid malignancies. Meanwhile, suspended single cells were also prepared by direct immunophenotyping with fluorescent conjugated antibody to CD3, CD5, CD7, CD19, CD20, CD56, CD57, CD38, CD138, CD22 on cell surface and CD3, CD22, Kappa, Lambda in cytoplasm. Immunophenotype of the gated lymphocytes were analyzed with multiple parameter cytometric analysis to come to a diagnosis. The accuracy and efficacy of the diagnosis was compared between methods of conventional and conventional combined with FCM immunophenotyping. Nine out of 38 lymph nodes failing to get a correct diagnosis by conventional methods were accurately diagnosed when FCM immunophenotyping was performed (P< 0.01), but there still remained 3 cases that did not get a clear pathological diagnosis. A total of 102 out of 104 peripheral blood or bone marrow samples were diagnosed according to the new WHO classification by flow cytometric immunophenotype analysis, compared to 90 out of 104 samples by conventional immnohistochemistry (P< 0.01). There remained 2 cases without clear diagnosis, including a patient with CD5 positive B cells. He was finally diagnosed as splenic marginal zone lymphoma, after cyclinD1 immunochemistry and FISH detection of translocation between chromosome 11 and 14 were performed to exclude mantle cell lymphoma. Multiple parameter phenotype analysis in cerebrospinal fluid by FCM was also informative for the diagnosis of the infiltration of leukemia or lymphoma cells in central nervous system. A patient with Burkit lymphoma was diagnosed as diffused infiltration of lymphoma cell in center nerves system after cerebrospinal fluid examination. The same patient was considered brain infection by CT scanning before FCM immunophenotype analysis was conducted. We concluded that multiple parameter phenotypic analysis by FCM can be used in the diagnosis, differentiated diagnosis and detection of minimal residue diseases in CLPDs. It provides a useful tool to improve the accuracy and efficacy of diagnosis in lymphoid malignancies.
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Lee, Hye Ryun, Inho Kim, Sung-Soo Yoon, Seonyang Park, Byoung Kook Kim, Hyun Kyung Kim, Han_Ik Cho, and Dong Soon Lee. "Usefulness of FICTION Method for the Evaluation of Response after Treatment in Multiple Myeloma." Blood 112, no. 11 (November 16, 2008): 5112. http://dx.doi.org/10.1182/blood.v112.11.5112.5112.
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Abstract Introduction: According to the new uniform response criteria of International Myeloma Working Group (IMWG), stringent complete response (sCR) is defined as a condition of normal free light chain (FLC) ratio and absence of clonal cells in the bone marrow (BM) by immunohistochemistry or immunofluorescence, in addition to the CR condition. The kappa/lambda ratio is assessed to identify clonal cells in the BM and a minimum of 100 plasma cells is required for analysis of clonal cells. However, the evaluation of kappa/lambda ratio by immunohistochemistry or immunofluorescence may be inaccurate, because it is difficult practically to countthe number of anti-kappa/lambda antibodies-stained cells in the BM section and in case of low percentage of residual plasma cells, flow cytometric evaluation is also difficult. To investigate whether FICTION (Fluorescence Immunophenotyping and interphase Cytogenetics as a Tool for the Investigation Of Neoplasms) technique can be used as a tools for evaluation of clonal cells after treatment in multiple myeloma, we performed FICTION on BM cells in follow-up patients with myeloma and compared the results of FICTION with other parameters. Method: 18 myeloma patients, whose BM examination and serum free light chain were checked at the same time after treatment, were enrolled in Seoul National University Hospital. We performed FICTION for the fluorescence in situ hybridization (FISH) items that were abnormal at initial BM examination of each patient. The selected probes were LSI 1q25/1p36/1p subtelomere probe (Vysis, Downers Grobe, IL, USA) for trisomy 1q25, LSI 13 (RB1) 13q14 probe (Vysis) for RB1 deletion, LSI IGH probe (Vysis) for IGH rearrangement and LSI p16 (9p21)/CEP 9 probe (Vysis) for p16 deletion. The FICTION results were reported by the percentage of plasma cells with abnormal FISH signals among plasma cells stained with anti-kappa and lambda antibodies labeled with fluorescein isothiocyanate (FITC). We compared FICTION results with response parameters, such as % plasma cells in BM aspirates, serum FLC ratio, M-component in serum and/or urine protein electrophoresis (PEP) and FISH results. Results: Among 18 patients in follow-up, 5 (28%) showed <5% plasma cells by light microscope based differential count in BM aspirates and normal FISH results below the cut-off level. However, these patients turned out to have clonal cells in BM by FICTION techniques. Among these 5 patients, 3 patients showed abnormal serum FLC ratio and the other 2 patients showed M-component in serum PEP with normal serum FLC ratio. One patient with plasma cells fewer than 5% in BM aspirates, no cytogenetic abnormality in FISH and no M-component in serum, showed abnormal serum FLC ratio and abnormal FICTION results; 2 (25%) of 8 plasma cells showed RB1 deletion in FICTION. After 2 years, this patient progressed to plasma cell leukemia. Conclusion: Results of FICTTON correlated with FLC ratio and/or M-component in serum, while the percentage of plasma cells in BM aspirates or FISH results did not. The assessment of percentage of plasma cells in bone marrow aspirates might be inaccurate due to poor aspiration technique including dilution and focal infiltration of plasma cells. Also, FISH results based on the percentage of abnormal cells among all bone marrow nucleated cells do not reflect clonal plasma cells. In clonclusion, we suggest that FICTION technique is more sensitive method for identification of residual malignant plasma cells with clonalityalong with the evaluation of response after treatment in multiple myeloma.
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Novick, Steven L., and John D. Baldeschwieler. "Fluorescence measurement of the kinetics of DNA injection by bacteriophage .lambda. into liposomes." Biochemistry 27, no. 20 (October 4, 1988): 7919–24. http://dx.doi.org/10.1021/bi00420a050.
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Atkins, C. G., and G. Hancock. "The 355 nm Photolysis of Methyl Nitrite." Laser Chemistry 9, no. 4-6 (January 1, 1988): 195–208. http://dx.doi.org/10.1155/lc.9.195.
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Single photon laser induced fluorescence (LIF) has been used to study the NO fragment produced in its ground electronic state from the 355 nm photolysis of CH3ONO. Populations of rotational, vibrational, spin-orbit and lambda doublet components have been measured. The results are in broad agreement with previous two photon LIF studies, and confirm the dynamics of the process in which the NO fragment departs from an essentially planar CONO framework with non-statistical energy partitioning in the internal states of the products.
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Dermietzel, R., A. Leibstein, W. Siffert, N. Zamboglou, and G. Gros. "A fast screening method for histochemical localization of carbonic anhydrase. Application to kidney, skeletal muscle, and thrombocytes." Journal of Histochemistry & Cytochemistry 33, no. 2 (February 1985): 93–98. http://dx.doi.org/10.1177/33.2.3918097.
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A simple method for histochemical localization of carbonic anhydrase using 5-dimethyl-amino-naphthalene-1-sulfonamide (DNSA) is described. Cryosections of tissues, or cell smears, are incubated in 3 to 10 X 10(-5) M DNSA and viewed in a fluorescence microscope. Upon excitation with ultraviolet light, sites of carbonic anhydrase localization can be identified by an intense blue fluorescence, which is due to the emission of blue light (lambda max = 470 nm) by carbonic anhydrase-DNSA complexes. This fluorescence can be largely suppressed by simultaneous incubation with 1 X 10(-4) to 2 X 10(-3) M concentrations of nonfluorescent carbonic anhydrase inhibitors, displacing DNSA from its binding site on the enzyme. Application of the method to kidney, skeletal muscle, and thrombocytes yields patterns of carbonic anhydrase localization that are in good agreement with results that have been obtained with a variety of other techniques.
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Zilberstein, D., J. Wilkes, H. Hirumi, and A. S. Peregrine. "Fluorescence analysis of the interaction of isometamidium with Trypanosoma congolense." Biochemical Journal 292, no. 1 (May 15, 1993): 31–35. http://dx.doi.org/10.1042/bj2920031.
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Isometamidium chloride (Samorin) is the only compound recommended for prophylaxis against bovine trypanosomiasis in sub-Saharan Africa. The fluorescence property of this compound was used to investigate the interaction of the molecule with in vitro-derived bloodstream forms of Trypanosoma congolense IL 1180. Incubation of isometamidium with trypanosomes at 37 degrees C for 180 min resulted in a gradual alteration of the lambda max. with time (from 600 to 584 nm) and an increase in the intensity of trypanosome-associated fluorescence of approx. 2-fold. The alteration in fluorescence was temperature-dependent and inhibited by the addition of N-ethylmaleimide. In contrast, with intact cells addition of digitonin caused a rapid increase in fluorescence intensity to approximately four times that observed with intact cells. Uptake of isometamidium was also determined using radiolabelled drug; the results indicated that the time course of the uptake process resembled the fluorescence profile and was temperature-dependent. The results therefore indicate that the alteration of fluorescence is due to interaction of isometamidium with an intracellular component(s) and that isometamidium is transported across the plasma membrane via a protein carrier. The data also indicate that the described fluorescence technique can be used to investigate the role of membrane transport in resistance to isometamidium.
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Wilcox, S. A., R. Toder, and J. W. Foster. "Rapid isolation of recombinant lambda phage DNA for use in fluorescence in situ hybridization." Chromosome Research 4, no. 5 (August 1996): 397–404. http://dx.doi.org/10.1007/bf02257276.
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Matsumoto, K., Y. Yamada, M. Takahashi, T. Todoroki, K. Mizoguchi, H. Misaki, and H. Yuki. "Fluorometric determination of carnitine in serum with immobilized carnitine dehydrogenase and diaphorase." Clinical Chemistry 36, no. 12 (December 1, 1990): 2072–76. http://dx.doi.org/10.1093/clinchem/36.12.2072.
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Abstract A fluorometric flow-injection method for determining carnitine with use of immobilized enzymes carnitine dehydrogenase (EC 1.1.1.108) and diaphorase (EC 1.8.1.4) was developed and applied to the assay of carnitine in serum of patients treated with valproic acid. After fractionation and hydrolysis of carnitines in serum samples by perchloric acid and potassium hydroxide, liberated carnitine was converted to resorufin by immobilized carnitine dehydrogenase and diaphorase in the presence of beta-NAD+ (1.0 mmol/L), resazurin (12.5 mumol/L), and Tris acetate (0.6 mol/L, pH 9.0) at 37 degrees C. The fluorescence intensity of resorufin was monitored at lambda Ex 560 nm and lambda Em 580 nm. The calibration curve was linear for carnitine amounts from 0.1 to 1.0 nmol. Quantitative analytical recovery and satisfactory within- and between-run imprecision of carnitine in each carnitine fraction were obtained. Interference by bilirubin, serum albumin, and hemoglobin was negligible. Carnitine deficiencies were detected in about 20% of the valproic acid-treated patients (n = 198). The present method should be useful for monitoring carnitine deficiencies in clinical laboratories.
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Sato, Masahiko, Janice Herring, John Kim, and Eli Lilly. "Reflected polarized darkfield imaging of bone surfaces." Proceedings, annual meeting, Electron Microscopy Society of America 50, no. 2 (August 1992): 956–57. http://dx.doi.org/10.1017/s0424820100129413.
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Reflected polarized light microscopy (Fig. 1A) was used previously to generate high contrast images of birefringent and light scattering samples, including bone surfaces and autoradiographic specimens. We now present a modification (Fig. 1B) of the Gullberg system with improved sensitivity for the characterization of bone specimens and quantitation of silver granules on autoradiographic specimens. Reflected imaging techniques were useful to generate high contrast images superior to transmitted light strategies, and both of the strategies presented can be adapted easily to any fluorescence microscope.Reflected light produced images free of refractile noise from materials through the thickness of the specimen which detracts from transmission darkfield imaging of silver grains and brightfield imaging of bone surfaces. The use of crossed polars also eliminated noise from stray light reflected off of internal microscope elements. The rotatable lambda/4 plate mounted on the objective front element (Fig. 1A) allowed considerable manipulation of image contrast, permitting dual imaging of silver granules, birefringent tissues in autoradiographic specimens and the surface topography of bone specimens by rotating the lambda/4 plate to 45°.
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Barrett, A. J., C. G. Knight, M. A. Brown, and U. Tisljar. "A continuous fluorimetric assay for clostridial collagenase and Pz-peptidase activity." Biochemical Journal 260, no. 1 (May 15, 1989): 259–63. http://dx.doi.org/10.1042/bj2600259.
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The peptide derivative N alpha-(2,4-dinitrophenyl)-L-prolyl-L-leucyl-glycyl-L-prolyl-L-tryptophanyl-D- lysine (Dnp-Pro-Leu-Gly-Pro-Trp-D-Lys) has been found to be a convenient substrate for the assay of clostridial collagenase and Pz-peptidase. The substrate shows a 25-fold enhancement of fluorescence (gamma ex. 283 nm, lambda em. 350 nm) following hydrolysis of the Leu2-Gly3 peptide bond. The value of Km for clostridial collagenase was 17 microM. The substrate for the first time makes possible continuous fluorimetric assays for Pz-peptidase and clostridial collagenase.
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Foskett, J. K. "Simultaneous Nomarski and fluorescence imaging during video microscopy of cells." American Journal of Physiology-Cell Physiology 255, no. 4 (October 1, 1988): C566—C571. http://dx.doi.org/10.1152/ajpcell.1988.255.4.c566.
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A video microscope designed to allow low light level fluorescence imaging of cells during simultaneous high-resolution differential interference contrast (DIC) imaging, without the fluorescence light losses of 60-90% normally associated with this contrast-enhancement technique, is described. Transmitted light for DIC imaging, filtered at greater than 620 nm, passes through standard DIC optical components, (1/4 lambda-plate, polarizer, and Wollaston prism) before illuminating the cells. Transmitted light and fluorescence emission pass through a second Wollaston prism but not through the analyzer, which is repositioned more distally in the optical path. Prisms designed to reflect light out a side port of the microscope to a video camera have been replaced with a dichroic mirror. This mirror reflects fluorescence emission out the side port to a low light-sensitive video camera. The spectrally distinct transmitted light continues through the dichroic mirror to an overhead camera through a polarizer (analyzer), which completes the DIC optical path. The fluorescence and DIC images can be viewed simultaneously on side-by-side video monitors, examined sequentially by an image-processing computer, or examined simultaneously using a video splitter/inserter. The ability to image cells with high resolution simultaneously with low light level fluorescence imaging should find wide applicability whenever it is necessary or desirable to correlate fluorescence intensity or distribution with specific cell structure or function.
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Rippin, J. J. "Analysis for Fully Oxidized Neopterin in Serum by High-Performance Liquid Chromatography." Clinical Chemistry 38, no. 9 (September 1, 1992): 1722–24. http://dx.doi.org/10.1093/clinchem/38.9.1722.
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Abstract Fully oxidized D-neopterin in serum can be measured by HPLC. Serum samples were preincubated with ferric nitrate/EDTA solution to remove any dihydroneopterin, which is unstable and may give spuriously high results because of its conversion to D-neopterin. Pterins were extracted onto solid-phase propylbenzenesulfonic acid minicolumns and eluted with a 1:5 (by vol) mixture of ammonia solution (308 g/L) in acetonitrile. Extracts were evaporated and then reconstituted in mobile phase (50 mL of methanol per liter of 50 mmol/L phosphate buffer, pH 6.2) before injection. Separation was performed with a 25-cm ODS2 column (particle size, 5 microns) at 32 degrees C with fluorescence detection (lambda ex 360 nm, lambda em 440 nm). The between-batch CV was 7.1% and 5.6% for neopterin concentrations of 21.7 and 67.3 nmol/L, respectively. The limit of detection was 0.75 nmol/L, and the mean recovery of the extraction procedure was 90% for neopterin and internal standard. Correlation with a radioimmunoassay (x) gave y = 0.99x + 0.64 (r = 0.970, Sy/x = 2.75). The method allows daily analysis of serum D-neopterin in small batches and is currently used to monitor patients undergoing bone-marrow transplant.
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Villablanca, J. G., J. M. Anderson, M. Moseley, C. L. Law, R. L. Elstrom, and T. W. LeBien. "Differentiation of normal human pre-B cells in vitro." Journal of Experimental Medicine 172, no. 1 (July 1, 1990): 325–34. http://dx.doi.org/10.1084/jem.172.1.325.
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The differentiation of surface Ig- pre-B cells into surface Ig+ B cells is a critical transition in mammalian B cell ontogeny. Elucidation of the growth factor requirements and differentiative potential of human pre-B cells has been hampered by the absence of a reproducible culture system that supports differentiation. Fluorescence-activated cell sorting and magnetic bead depletion were used to purify fetal bone marrow CD10+/surface mu- cells, which contain 60-70% cytoplasmic mu+ pre-B cells. CD10+/surface mu- cells cultured for 2 d were observed to differentiate into surface mu+ cells. Analysis by Southern blotting provided direct evidence that rearrangement of kappa light chain genes occurs in culture, and flow cytometric analysis revealed the appearance of surface Ig+ B cells expressing mu/kappa or mu/lambda. Unexpectedly, the kappa/lambda ratio in differentiated cells was the inverse of what is normally observed in adult peripheral blood. Differentiation occurs in the absence of exogenous growth factors or cytokines, suggesting that a stimulus-independent differentiative inertia might characterize pre-B cells in vivo. Future use of this model will facilitate our understanding of normal and abnormal human pre-B cell differentiation.
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Teissier, E., E. Walters-Laporte, C. Duhem, G. Luc, J. C. Fruchart, and P. Duriez. "Rapid quantification of alpha-tocopherol in plasma and low- and high-density lipoproteins." Clinical Chemistry 42, no. 3 (March 1, 1996): 430–35. http://dx.doi.org/10.1093/clinchem/42.3.430.
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Abstract We have developed two methods for measuring the alpha-tocopherol content in plasma and lipoproteins (LDL and HDL). In procedure 1, plasma or lipoproteins are deproteinized with ethanol containing delta-tocopherol as internal standard and then extracted with hexane or ethyl acetate. The organic layer is removed and evaporated, and the residue is redissolved in methanol and injected into a reversed-phase HPLC. In procedure 2, plasma or lipoproteins are diluted in a methanol and ethanol mixture containing the same internal standard. The solution is vortex-mixed, centrifuged, and directly injected into the column. The tocopherols are eluted with an isocratic methanol mobile phase at a flow rate of 1 mL/min and detected by fluorescence (lambda(exc)= 295 nm, lambda(em)= 330nm). Recoveries are approximately 100% in both cases. Between-run CVs were 8.39% for procedure 1 and 6.55% for procedure 2. Small sample requirement, simplicity of sample preparation, short assay time, and good reproducibility make procedure 2 ideal for clinical or research use. This method was applied to determination of alpha-tocopherol in plasma of patients whose diet was supplemented with alpha-tocopherol and in LDL and HDL.
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Wong, G. Y., D. Gebhard, A. Mittleman, M. Hancu, and B. Koziner. "Analysis of cell surface light chain immunoglobulin expression by flow cytometry in normal controls: a new mathematical approach." Journal of Histochemistry & Cytochemistry 33, no. 2 (February 1985): 119–26. http://dx.doi.org/10.1177/33.2.3918096.
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It has been observed in flow cytometric studies that in normal individuals there are proportionally more kappa+ than lambda+ bearing light chain B-cells. Overt predominance of one type of light chain bearing cell over the other is a characteristic of B-cell neoplasias, a phenomenon called clonal excess (CE). A mathematical model using the Weibull distribution is proposed for studying such an excess. The new approach is desirable for two reasons: First, it is parametric and hence offers a more sensitive and versatile analysis than its nonparametric counterparts. Second, it utilizes only the relevant information from the upper tails of the distributions of the fluorescence intensity of the kappa+ and lambda+ cells. Two measures of CE based on the Weibull model are proposed, and a normal range of variability was determined for each measure using a random sample of 48 normal controls. Such normal ranges are particularly useful in detecting cancer patients with minimal B-cell neoplasias. A comparative study of the new measures, Ault's maximum difference measure, and a measure based on Ligler's method showed that the parametric approach provides much more sensitivity than both the nonparametric ones.