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PubMed2006

Capture of extracellular zinc ions by astrocytes.

Synaptic zinc ions released during synaptic transmission interact with pre- and postsynaptic neuroreceptors, thus modulating neurotransmission. It is likely that they have to be efficiently cleared from the extracellular milieu to assure subsequent synaptic events. Both neurons and glia are assumed to participate in this clearance by mechanisms that are not fully understood. In this study, electron microscopic zinc cytochemistry has shown zinc-electrondense particles associated with hippocampal astrocytic membranes frequently found accumulated in stacked lamellae. In cultured astrocytes, the use of zinc fluorochromes and endocytic markers allowed the simultaneous imaging of the capture of extracellular zinc simultaneously to plasma membrane markers; this endocytic process was inhibited by high sucrose concentrations. Finally, electron microscopy of zinc-loaded and fluorochrome photoconverted cells demonstrated some early events of extracellular zinc capture as well as its late accumulation in lysosome-like organelles.

Animals↗

PubMed2002

Evidence of high incidence of EGFRvIII expression and coexpression with EGFR in human invasive breast cancer by laser capture microdissection and immunohistochemical analysis.

EGFRvIII was first reported in human glioblastomas. Subsequent reports indicated EGFRvIII protein to be frequently detected in several other human cancers, but not in normal tissues. Our previous studies suggested that EGFRvIII could induce a transformation from ligand-dependent non-tumorigenic cell line to ligand-independent malignant phenotype cells in vitro and in vivo. Transfection of EGFRvIII in MCF-7 cell line resulted in a 3-fold increase in colony formation and significantly enhanced tumorigenicity in nude mice (p < 0.001). EGFRvIII could also induce ErbB-2 phosphorylation. The existence and significance of EGFRvIII transcript in human breast cancer, however, was not reported. In our study, we detected the presence of EGFRvIII mRNA and revealed a high incidence (67.8%) of EGFRvIII transcript in human primary invasive breast cancer by utilizing laser capture microdissection (LCM)/RT-PCR to capture pure breast cancer cells. In addition, 57.1% of the infiltrating breast carcinomas expressed both EGFRwt and EGFRvIII mRNA in the same tumor. There is no detectable EGFRvIII mRNA in normal breast tissue. Evaluation of the EGFRwt and EGFRvIII protein levels in the same sample sets by immunohistochemical analysis further confirmed the LCM/RT-PCR finding. Our study provides first direct evidence of high incidence of coexpression of EGFRvIII and EGFRwt in human invasive breast cancer tissue. The unique characteristics and high prevalence of EGFRvIII in invasive human breast cancer as well as negative expression in normal breast may suggest its important role in breast carcinogenesis and make it an ideally potential target for treatment of breast cancer without interrupting normal EGFR signaling.

Adult↗

PubMed2005

p53 mutation profiling of multiple esophageal carcinoma using laser capture microdissection to demonstrate field carcinogenesis.

Inactivation of the p53 tumor suppressor gene is one of the most frequent genetic alterations observed in human esophageal carcinomas. In patients with esophageal carcinoma, one of the significant pathological features of the tumor is the presence of multiple lesions within the esophagus. However, the molecular mechanisms involved in the occurrence of multiple lesions have remained elusive. To characterize p53 alterations in multiple esophageal carcinomas and to study their roles in carcinogenesis, we performed p53 immunohistochemical and p53 mutation analyses using laser capture microdissection on surgically resected human esophageal carcinomas from 11 patients: 9 patients with multiple esophageal carcinomas, 1 with an intramural metastasis lesion within the esophagus and 1 with an intraepithelial carcinoma lesion contiguous to the main lesion. In each of the patients with multiple esophageal carcinomas, we examined samples from 1 main lesion and 1 representative concomitant lesion. Molecular analyses of samples from fresh-frozen normal tissues and tumor tissues of the main lesion (whole tumor) were also performed by the same method. p53 protein accumulation was observed in 16 (72.7%) of 22 lesions from the 11 cases. No p53 mutation was found in normal esophageal tissues. In the 9 cases of multiple esophageal carcinomas, point mutations were detected in the whole tumor in 1 (11.1%) case, in the microdissected tumor samples of main lesions in 3 (33.3%) cases and in the microdissected tumor samples of concomitant lesions in 3 (33.3%) cases. For the microdissected tumor samples, there was a 54.5% (12/22) concordance rate between the results of immunostaining and molecular analysis. In the 8 cases of whole tumors in which a p53 mutation was not observed, 2 cases revealed p53 mutation in the microdissected tumor samples of the main lesion. All 6 cases of multiple esophageal carcinomas that showed a p53 mutation in the microdissected tumor sample had a discordant p53 mutational status between the main and concomitant lesions. In contrast, both the intramural metastasis lesion and the intraepithelial carcinoma contiguous to the main lesion showed p53 mutational patterns identical to those of the main lesions. In conclusion, the analysis of microdissected DNA by laser capture microdissection is useful for characterizing the heterogeneity of the p53 gene mutation in multiple carcinoma lesions that cannot be accurately analyzed in whole esophageal tumor DNA. The finding of different p53 gene mutations among multiple esophageal carcinoma lesions suggest further evidence of multicentric or field carcinogenesis of the human esophagus.

Carcinoma in Situ↗

PubMed2007

Free energy profiles for monomer capture in Grubbs- and SHOP-type olefin polymerization catalysts: a constraint ab initio molecular dynamics study.

Density functional theory together with Car-Parrinello ab initio molecular dynamics simulation has been used to investigate the free energy profiles (FEP) of monomer capture in Grubbs- and SHOP-type olefin polymerization catalysts. The FEPs along the reaction coordinates at 300 K were determined directly by a point wise thermodynamic integration technique. Comparison between potential energy profile (PEP) and the FEP has been made. The results show that, for both catalysts, the PEP for the monomer ethylene uptake by the metal center is a typical Morse curve without energy barrier. However, a small barrier (1.8 kcal/mol for Grubbs catalyst and 2.4 kcal/mol for SHOP catalyst) exists on the FEP. The pi complexation energy on the FES at 300 K is higher by 10-12 kcal/mol over that on the PES. The differences between FES and PES are due to entropy contribution. Slow growth simulations on the ethylene capture process show that the ethylene attacks the metal center by an asynchronous mode. This indicates that the forming of the pi-bonding between the metal and ethylene is initiated by electrophilic attack of the metal to one of the ethylene carbons.

Alkenes↗

PubMed1994

Small cardioactive peptide B increases the responsiveness of the neural system underlying prey capture reactions in the pteropod mollusc, Clione limacina.

Effects of small cardioactive peptide B (SCPB) on cerebral neurons which underlie prey capture in the carnivorous pteropod mollusc, Clione limacina, were investigated. SCPB in concentrations of 10 microM and higher produced direct activation of cerebral ganglion neurons underlying extrusion of buccal cones used in prey capture. SCPB in lower concentrations, between 1 and 5 microM, did not have a noticeable effect on the membrane potentials of these neurons; however, it significantly increased their responsiveness to sensory inputs from the tactile stimulation of the head, and their ability to generate afterdischarge activity. SCPB immunoreactivity was observed in cell bodies in buccal, cerebral, pedal, and intestinal ganglia, as well as in the anterior esophagus and in buccal cones where fibers stained intensely. These electrophysiological and immunohistochemical data suggest that SCPB may have a physiological role in feeding arousal in Clione.

Action Potentials↗

PubMed2003

Functional morphology of prey capture in the sturgeon, Scaphirhynchus albus.

Acipenseriformes (sturgeon and paddlefish) are basal actinopterygians with a highly derived cranial morphology that is characterized by an anatomical independence of the jaws from the neurocranium. We examined the morphological and kinematic basis of prey capture in the Acipenseriform fish Scaphirhynchus albus, the pallid sturgeon. Feeding pallid sturgeon were filmed in lateral and ventral views and movement of cranial elements was measured from video sequences. Sturgeon feed by creating an anterior to posterior wave of cranial expansion resulting in prey movement through the mouth. The kinematics of S. albus resemble those of other aquatic vertebrates: maximum hyoid depression follows maximum gape by an average of 15 ms and maximum opercular abduction follows maximum hyoid depression by an average of 57 ms. Neurocranial rotation was not a part of prey capture kinematics in S. albus, but was observed in another sturgeon species, Acipenser medirostris. Acipenseriformes have a novel jaw protrusion mechanism, which converts rostral rotation of the hyomandibula into ventral protrusion of the jaw joint. The relationship between jaw protrusion and jaw opening in sturgeon typically resembles that of elasmobranchs, with peak upper jaw protrusion occurring after peak gape.

Animals↗

PubMed1985

Aquatic prey capture in ambystomatid salamanders: patterns of variation in muscle activity.

Functional morphologists commonly study feeding behavior in vertebrates by recording electrical activity from head muscles during unrestrained prey capture. Rarely are experiments designed to permit a partitioning variation in muscle electrical activity patterns. Analysis of muscle activity during aquatic prey capture in two morphologically distinct species of salamanders, Ambystoma dumerilii and A. mexicanum, is conducted to assess variation at four levels: between species, among individuals within species, among experiments conducted on different days, and among feedings. The results show that 1) mean correlations among the 11 electromyographic variables measured for each feeding are low and vary considerably among individuals, 2) many of the variables show significant differences among experimental days, 3) only one variable, the difference in timing between the depressor mandibulae and sternohyoideus muscles, showed significant variation between species, and 4) seven of the 11 variables showed significant variation among individuals within species. Overall, the variation between feedings (trials) was high, and there was some variation between days on which the experiments were conducted. Neither electrode position within the muscle nor satiation contributed to the high trial variance. The results suggest that functional analyses of feeding behavior should include an assessment of variation due to individuals, days, and trials, because the amount of variation at these levels may render differences between species nonsignificant.

Activity Cycles↗

PubMed2002

Mass spectrometry in coupling with affinity capture-release and isotope-coded affinity tags for quantitative protein analysis.

Affinity capture-release electrospray ionization mass spectrometry (ACESIMS) and isotope-coded affinity tags (ICAT) are two recently introduced techniques for the quantitation of protein activity and content with applications to clinical enzymology and functional proteomics, respectively. One common feature of these methods is that they use biotinylated tags that function as molecular handles for highly selective and reversible affinity capture of conjugates from complex biological mixtures such as cell homogenates and sub-cellular organelles. ACESIMS uses synthetic substrate conjugates specifically to target cellular enzymes that, when deficient, are the cause of genetic diseases. Multiplex determination of enzyme activities is used for the diagnosis of lysosomal storage diseases. The ICAT method relies on selective conjugation of cysteine thiol groups in proteins, followed by enzymatic digestion and quantitative analysis of peptide conjugates by mass spectrometry. Another common feature of the ACESIMS and ICAT approaches is that both use conjugates labeled with stable heavy isotopes as internal standards for quantitation. Selected applications of the ACESIMS and ICAT techniques are presented that include molecular-level diagnosis of genetic diseases in children and quantitative determination of protein expression in cells.

Affinity Labels↗

PubMed2003

Mucosal immunization with inactivated HIV-1-capturing nanospheres induces a significant HIV-1-specific vaginal antibody response in mice.

Mucosal secretory IgA is considered to have an important role in the prevention of human immunodeficiency virus type 1 (HIV-1) transmission through sexual intercourse. Therefore, substances that induce HIV-1-specific IgA antibody in the genital tract may become promising candidates for prophylactic vaccine against HIV-1 infection. We have previously reported that concanavalin A-immobilized polystyrene nanospheres (Con A-NS) could efficiently capture HIV-1 particles and gp120 antigens on their surface and that intravaginal immunization with inactivated HIV-1-capturing nanospheres (HIV-NS) induced vaginal anti-HIV-1 IgA antibody in mice. In this study, various strategies for immunization with HIV-NS were undertaken to induce HIV-1-specific IgA response in the mouse genital tract. HIV-NS were administered intravaginally, orally, intranasally or intraperitoneally to mice. Progesterone treatment enhanced the anti-HIV-1 IgA response to intravaginal immunization significantly, but intranasal immunization with HIV-NS was more effective compared with other immunization routes in terms of vaginal IgA response. In addition, vaginal washes from intranasally immunized mice were capable of neutralizing HIV-1(IIIB). Thus, application of HIV-NS is a practical approach to promote HIV-1-specific IgA response by the vaginal mucosa in the mouse and intranasal appears to be an effective immunization route in this animal model. Intranasal immunization with HIV-NS should be further pursued for its potential as an HIV-1 prophylactic vaccine.

AIDS Vaccines↗

PubMed1992

Hepatitis C virus antibody detection by a line immunoassay and (near) full length genomic RNA detection by a new RNA-capture polymerase chain reaction.

A rapid and simple RNA-capture polymerase chain reaction assay (RCPA) for detection of hepatitis C virus (HCV) is described. The assay detects specifically the presence of (near) full length genomic RNA of HCV by capturing HCV-RNA at the 3' terminal end on magnetic beads, followed by cDNA synthesis and PCR with 5' end specific primers. Sera were obtained from 30 chimpanzees inoculated with non-A, non-B hepatitis material from various sources, 28-122 months after infection. The sera were tested for the presence of HCV-RNA by RCPA and for HCV antibodies by a Line ImmunoAssay (Inno-LIA HCV Ab). Both tests were compared and show a high degree of agreement. Screening of 30 chimpanzee sera revealed either clearing of the virus below detection level (22/30) or development of a HCV carrier state (8/30). Only 1 of 11 LIA-indeterminate samples was positive by RCPA. As the RCPA is more sensitive, it can be used to test for the presence of HCV in sera which are classified indeterminate by the LIA. The outcome of the infection seems to be independent of the nature of the inocula, suggesting that the individual immune response could determine either clearing of the virus or the development of chronic infection.

Animals↗

PubMed1977

Electron-capture GLC determination of nanogram to picogram amounts of isosorbide dinitrate.

A GLC method for the determination of plasma isosorbide dinitrate using electron-capture detection is described. The organic nitrates are especially suited for electron-capture detection if the detector temperature is optimized for maximum sensitivity, e.g., 175 degrees. Proper maintenance of the detector and column assures reproducible data in the low nanogram range. The extraction procedure described is simple, efficient, and expedient for processing large numbers of samples. The method was used to study plasma levels in four human volunteers after a single dose of a 5-mg chewable isosorbide dinitrate tablet. Concentration levels of isosorbide dinitrate as low as 0.5 ng/ml of plasma can be measured by this procedure.

Adult↗

PubMed1979

Improved electron-capture GLC determination of alprenolol and oxprenolol in serum using a wall-coated open tubular column.

An accurate, rapid, and sensitive GLC determination of alprenolol and oxprenolol in serum is described. This method combines an electron-capture detector with a wall-coated open tubular column. The lowest detectable amount of the halogenated (heptafluoroacyl) derivatives of alprenolol and oxprenolol is 2 pg. The high resolving power and the rapid elution time of the wall-coated open tubular column dramatically improve the performance (accuracy and sensitivity) of the conventional electron-capture detector and packed column system in these determinations. This method allows the use of small volumes of sample (100-200 microliter), does not require redistilled reagents, and has a simplified extraction procedure.

Alprenolol↗

PubMed1979

Simple and specific electron-capture GLC assay for plasma and urine ephedrine concentrations following single doses.

An electron-capture GLC procedure for determination of plasma ephedrine concentrations is described. The procedure is capable of determining 2 ng/ml of ephedrine and is adequate for following profiles after 25-mg single doses. Pentane extraction of the drug and the internal standard and formation of the N-pentafluorobenzoyl derivatives were followed by GLC. Plasma ephedrine concentrations following a 24-mg dose of ephedrine hydrochloride to a human volunteer are presented. Formation of N-trifluoroacetyl, N-pentafluoropropionyl, N-heptafluorobutryl, and N-pentafluorobenzoyl derivatives and their GLC-mass spectrometric identification are discussed together with comparative electron-capture sensitivities of these derivatives toward a nickel-63 detector. The detection of the N-pentafluorobenzoyl derivative of ephedrine is at least 100-fold greater in sensitivity than detection of the N-trifluoroacetyl derivative.

Chromatography, Gas↗

PubMed1980

Determination of pindolol in biological fluids by electron-capture GLC.

An electron-capture GLC method was developed for measuring pindolol in human plasma and urine. The unchanged drug was extracted with benzene from alkalinized plasma or urine using propranolol as the internal standard. Both compounds subsequently were back-extracted into 0.1 M HCl and then into benzene. After evaporation of the organic phase, the compounds were derivatized with trifluoroacetylimidazole to form the trifluoroacetyl ester of pindolol and propranolol. These derivatives then were analyzed by electron-capture GLC. The method allowed the measurement of concentrations as low as 1 ng of pindolol/ml of plasma and was applied successfully to determinations of plasma levels in humans after oral administraton of a single 10-mg dose of pindolol.

Chemical Phenomena↗

PubMedDate not supplied

The role of electron capture dissociation in biomolecular analysis.

The introduction of electron capture dissociation (ECD) to electrospray (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) constitutes a significant advance in the structural analysis of biomolecules. The fundamental features and benefits of ECD are discussed in this review. ECD is currently unique to FT-ICR MS and the fundamentals of that technique are outlined. The advantages and complementarity of ECD in relation to other tandem mass spectrometry (MS/MS) techniques, such as infrared multiphoton dissociation (IRMPD) and sustained off-resonance collision-induced dissociation (SORI-CID), are discussed. The instrumental considerations associated with implementation of ECD, including activated ion techniques and coupling to on-line separation techniques, are covered, as are the allied processes electronic excitation dissociation (EED), electron detachment dissociation (EDD), and hot electron capture (HECD). A major theme of this review is the role of ECD in proteomics, particularly for characterization of post-translational modifications (phosphorylation, glycosylation, carboxyglutamic acid, sulfation, acylation, and methionine oxidation) and the top-down approach to protein identification. The application of ECD to the analysis of polymers, peptide nucleic acids, and oligonucleotides is also discussed.

Cyclotrons↗

PubMed2001

Use of laser capture microdissection, cDNA microarrays, and tissue microarrays in advancing our understanding of prostate cancer.

One difficulty in studying epithelial tumors has been the inability to isolate pure samples for DNA and RNA analysis. Prostate cancer, with its infiltrative nature, is particularly challenging. The Combination of several new technologies should help overcome these hurdles. Laser capture microdissection uses a laser beam to achieve transfer of pure cell populations for isolation of DNA, RNA, and protein. High-throughput analysis of these samples can be achieved by using cDNA expression microarrays. High-density tissue microarrays should allow for validation of differentially expressed genes. This review will concentrate on the application of laser capture microdissection, cDNA microarrays, and tissue microarrays in the area of prostate cancer research.

Breast Neoplasms↗

PubMed2006

Development of a "reverse capture" autoantibody microarray for studies of antigen-autoantibody profiling.

Diagnosing cancers based on serum profiling is a particularly attractive concept. However, the technical challenges to analysis of the serum proteome arise from the dynamic range of protein amounts. Cancer sera contain antibodies that react with a unique group of autologous cellular antigens, which affords a dramatic amplification of signal in the form of antibodies relative to the amount of the corresponding antigens. The serum autoantibody repertoire from cancer patients might, therefore, be exploited for antigen-antibody profiling. To date, studies of antigen-antibody reactivity using microarrays have relied on recombinant proteins or synthetic peptides as arrayed features. However, recombinant proteins and/or synthetic peptides may fail to accurately detect autoantibody binding due to the lack of proper PTMs. Here we describe the development and use of a "reverse capture" autoantibody microarray. Our "reverse capture" autoantibody microarray is based on the dual-antibody sandwich immunoassay platform of ELISA, which allows the antigens to be immobilized in their native configuration. As "proof-of-principle", we demonstrate its use for antigen-autoantibody profiling with sera from patients with prostate cancer and benign prostate hyperplasia.

Aged↗

PubMed2003

Determination of coplanar and non-coplanar polychlorinated biphenyls in human serum by gas chromatography with mass spectrometric detection: electron impact or electron-capture negative ionization?

A time- and cost-saving method for the congener-specific analysis of polychlorinated biphenyls (PCBs) in human serum has been developed and validated. After two fast extraction and clean-up steps, analyses were performed using gas chromatography coupled with mass spectrometry with single ion monitoring (GC/SIM-MS), either in electron impact (EI) or electron-capture negative ionization (ECNI) mode. For the determination of dioxin-like congeners, an improvement in EI-MS sensitivity is desirable and use of NI is thus preferred. The procedure was validated for 12 dioxin-like congeners by analyzing spiked samples on three different days and using (13)C(12)-labelled analogues as internal standards. When using an NCI source, the limit of quantification was assessed at 0.01 microg/L, except for PCBs #77 and #81, which cannot be reliably detected below 0.05 microg/L. For the lower chlorinated non-dioxin-like congeners, NI offers less selectivity because of limited fragmentation. Electron impact ionization and electron-capture negative ionization mode can therefore be considered to be complementary for the determination of PCB congeners in the general population.

Dioxins↗