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PubMed1990

Detection of measles virus genomic sequences in SSPE brain tissue by the polymerase chain reaction.

The polymerase chain reaction (PCR) was modified to detect RNA genomic sequences by generating cDNA copies of these sequences as a preliminary step. Oligonucleotide primer pairs complementary to sequences in each of the five major structural protein genes of the measles virus (nucleocapsid protein, phosphoprotein, matrix protein, fusion protein, and hemagglutinin protein) were synthesized. PCR products were tentatively identified by visualization of bands of the appropriate size by ethidium bromide staining after gel electrophoresis, and identity was confirmed by subsequent restriction enzyme cleavage of the products at predetermined sites to yield fragments of predicted size. This method successfully amplified 400-500 base regions from each of these five genes in RNA extracts of wild measles virus cultured in Vero cells and in RNA extracted from most of the SSPE brain tissues tested, but not in RNA from any control brain tissues. Measles virus genome was detected in SSPE brain tissues stored frozen for as long as 27 years and formalin-fixed paraffin-embedded subacute sclerosing panencephalitis (SSPE) brain tissues as old as 9 years. This method provides a simple, rapid and highly sensitive means of detecting and identifying sequences of RNA genomes by PCR. The success of this method in detecting measles virus in SSPE brain tissue suggests that PCR is appropriate to investigate the possible presence of RNA viruses in other neurological disorders of unknown etiology.

Adolescent↗

PubMed2004

Adult sclerosing rhabdomyosarcoma: cytogenetic link with embryonal rhabdomyosarcoma.

Rhabdomyosarcomas are classified into three well-defined categories: embryonal, alveolar and pleomorphic rhabdomyosarcoma. Recently, seven cases of an unusual adult type of rhabdomyosarcoma with a prominent hyaline sclerosis have been described. We report the hitherto unreported cytogenetic changes of an adult sclerosing rhabdomyosarcoma. A 79-year-old woman underwent an amputation for a rapidly growing soft tissue mass in the anterior compartment of the right lower leg. The tumor infiltrated the tibia. On histology, a fascicular spindle to round cell proliferation, embedded in a prominent hyaline matrix, was seen. Immunohistochemistry showed focal desmin, myogenin and MyOD1 expression, and electron microscopy revealed Z-band material. Cytogenetic analysis disclosed a 44-49,XX,+del(1)(p22)[2],+11,+16[5],+18[12],+21[3],-22 [cp13] karyotype. Using fluorescent in situ hybridization (FISH) analysis, the tumor cells were negative for FOXO1A-disrupting translocations specific for alveolar rhabdomyosarcoma. The chromosomal composition of malignant cells resembled the pattern of numerical changes frequently observed in embryonal rhabdomyosarcoma, suggesting a close relationship of an adult sclerosing rhabdomyosarcoma with this entity.

Aged↗

PubMed1991

Protease nexin-1 activity in cultured Schwann cells.

We report that protease nexin-1 (PN-1), a serine protease inhibitor known to have neurite-promoting effects, is made by Schwann cells in tissue culture. Three modalities have been used to demonstrate the presence of PN-1 in Schwann cell cultures. Immunostaining of the cultures with anti-PN-1 antibody gives positive staining over cells and matrix. Western blots of Schwann cell conditioned medium (CM) using anti-PN-1 antibody show a band that co-migrates with the PN-1 standard at 45 kDa. Biochemical assay for protease inhibitory activity shows that CM inhibits thrombin activity in a calorimetric assay. The CM-mediated inhibition of thrombin is reversed if the CM is pre-incubated with anti-PN-1 antibody.

Amyloid beta-Protein Precursor↗

PubMed1997

Interpretation of multiple Q(0,0) bands in the absorption spectrum of Mg-mesoporphyrin embedded in horseradish peroxidase.

Mg-mesoporphyrin horseradish peroxidase (MgMP-HRP) and MgMP-HRP complexed with naphtohydroxamic acid (NHA) have been studied by fluorescence line narrowing (FLN) and pressure tuning spectral hole burning (SHB) techniques. In each sample, the low temperature absorption spectra show more than one transition in the origin range of the Q band. Comparisons with broad-band fluorescence spectra and FLN studies suggest that the multiple band feature originates from the presence of different configurations of the metal-porphyrin that are subject to Qx-Qy splitting within the protein cavity. This suggestion is supported by pressure tuning SHB studies. In the uncomplexed as well as in the NHA-complexed form of MgMP-HRP, irradiation in the Q band produces photoproduct bands, which has been attributed to a species with smaller Qx-Qy splitting. In an amorphous matrix, on the other hand, only one form of MgMP could be found, and no splitting could be observed. The binding of NHA does not significantly alter the bulk parameters of the protein matrix, but it reduces the structural variety in the configuration of MgMP to a single form with a more distorted structure and thus with an enlarged Qx-Qy splitting.

Horseradish Peroxidase↗

PubMed2002

Experimental verification of the effects of optical wavelength on the amplitude of laser generated ultrasound in polymer-matrix composites.

Laser ultrasound is now integrated into the manufacturing process of some of the most modern aircraft for the inspection of composite parts. Unfortunately, for some material and process combinations, laser-ultrasound suffers from a lack of sensitivity. In laser-ultrasound generation, optical penetration depth plays a very important role. It was shown that changing the generation wavelength from the 10.6 microm of the CO2 laser to the 3-4 microm range can significantly improve generation efficiency. In this paper, ultrasonic displacements are compared to measurements of optical penetration depth in different polymer-matrix composites. Ultrasonic waves were generated using an optical parametric oscillator operating in the 3.0-3.5 microm band and optical penetration depth spectra were evaluated using quantitative photoacoustic spectroscopy. The relative amplitudes of the generated ultrasonic waves track closely the optical penetration depth spectra. These results experimentally demonstrate the importance of optical penetration in the laser-ultrasound generation process.

Journal Article↗

PubMed2001

Application of concentrated TiO2 sols for gamma-ray radiation dosimetry.

Upon exposure to gamma-radiation, a concentrated TiO2 sol changes from colorless to deep blue with an absorption maximum at 540 nm. The absorption has been assigned to trapped electrons or Ti3+ states in the solid matrix based on its spectroscopic similarity to the samples irradiated with UV light. Unlike the conduction-band electrons generated from direct excitation by UV radiation, the origin of the trapped electrons during gamma-ray irradiation may be traced to a series of reducing species produced by the high energy electrons, which in turn, are the direct result of gamma-irradiation. As the absorption intensity is linearly related to the duration of exposure to gamma-radiation, it may have an application in gamma-ray dosimetry. The sensitivity of its dosage response has been found to be influenced by the semiconductor particle concentration and the dispersing solvent.

Journal Article↗

PubMed2003

Perfusion chromatography purification of a 15 kDa rice prolamin.

Prolamin extracted from rice flour using 55% n-propanol contained protein impurities. Reverse phase high-performance liquid chromatography (HPLC) on a perfusion column R2/H was used to separate rice prolamin from other proteins in less than 5 min. Prolamin eluted as the major peak. The isolated prolamin migrated as a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis using a 4-12% Bis-Tris gel. Matrix-assisted laser desorption ionization mass spectrometry identified the rice prolamin as a 15 013 Da protein. The surface hydrophobicity (S(o)) of the HPLC-separated protein fractions was measured using the hydrophobic fluorescent probe PRODAN. A comparison was made with the surface hydrophobicity (S(o)) of corn prolamin and bovine serum albumin. Surface hydrophobicity values and solubility in 90% ethanol assisted in rice prolamin identification from other chromatographic peaks. The advantage of perfusion chromatography in purifying rice prolamin from other rice proteins included the reduced separation time, the speed at which the separation was carried, and the ability to regenerate the column in a short period of time and allow for more samples to be purified and separated.

Chemical Phenomena↗

PubMed2006

3Sigma- -X 3Sigma- electronic transition of linear C6H+ and C8H+ in neon matrixes.

The electronic absorption spectra of linear C6H+ and C8H+ were recorded in 6 K neon matrixes following mass selective deposition. The (1) 3Sigma- -X 3Sigma- electronic transition is identified with the origin band at 515.8 and 628.4 nm for l-C6H+ and l-C8H+, respectively. One strong (near 267 nm) and several weaker electronic transitions of l-C8H+ have also been observed in the UV. The results of ab initio calculations carried out for linear and cyclic C6H+ are consistent with the assignment.

Journal Article↗

PubMed2006

Lifetime determination of fluorescence and phosphorescence of a series of oligofluorenes.

The photoluminescence (PL) properties of oligofluorenes with 2-ethylhexyl group in 9, 9' position in solution and as thin films were investigated by time-resolved techniques at both room temperature and 77 K. The fluorescence lifetimes of the oligomers decrease with chain length. The lifetimes tau follow the relation tau=386+808(1/n) (ps) where n is the number of fluorene units in the oligomer. Concentration and laser excitation energy dependences of PL spectra of the oligofluorenes are also given. Phosphorescence was observed for oligofluorenes in the frozen matrix of MTHF at 77 K. The lifetime of phosphorescence increases with increasing molecular length. Similar emission bands were observed for oligofluorenes with a central ketogroup. A lifetime analysis clearly reveals that the "green emission" of the oligomers free of ketogroups results from a phosphorescence with lifetime tau of 3 ms while the green emission from the keto-oligomer is a fluorescence from a charge transfer pi-pi* level of tau=8 ns.

Journal Article↗

PubMed2006

P-selectin mediates metastatic progression through binding to sulfatides on tumor cells.

Hematogenous carcinoma metastasis is associated with tumor cell emboli formation, which is now known to be facilitated by selectins. P-selectin-mediated interactions of platelets with cancer cells are based mostly on mucin- and glycosaminoglycan-type selectin ligands. We previously showed that mouse colon carcinoma cells (MC-38) carry P-selectin ligands of nonmucin origin, which were not identified. Here we show that P-selectin ligands recognized on MC-38 cells are sulfated glycolipids, thereby facilitating experimental metastasis in a syngeneic mouse model. Metabolic inhibition of sulfation by incubation of cells with sodium chlorate almost completely abrogated P-selectin binding. Metabolic labeling of MC-38 cells with (35)S sulfate revealed only a single band as detected by high-performance thin layer chromatography analysis of a total lipid extract. Matrix-assisted laser desorption/ionization tandem time-of-flight/time-of-flight analysis (MALDI-TOF-TOF) analysis of the purified sulfate-containing lipid fraction identified the selectin ligand to be a sulfated galactosylceramide SM4 (HSO(3)-3Galbeta-1Cer). Modulation of glycolipid biosynthesis in MC-38 cells altered P-selectin binding, thereby confirming sulfoglycolipids to be major P-selectin ligands. In addition, P-selectin was also found to recognize lactosylceramide sulfate SM3 (HSO(3)-3Galbeta-4Glcbeta-1Cer) and gangliotriaosylceramide sulfate SM2 [GalNAcbeta-4(HSO(3)-3)Galbeta-4Glcbeta-1Cer] in human hepatoma cells. Finally, the enzymatic removal of sulfation from the cell surface of MC-38 cells resulted in decreased P-selectin binding and led to attenuation of metastasis. Thus, SM4 sulfatide serves as a native ligand for P-selectin contributing to cell-cell interactions and to facilitation of metastasis.

Animals↗

PubMed2006

Anomalous isomeric decays in 174Lu as a probe of K mixing and interactions in deformed nuclei.

A K(pi)=13+, 280 ns four-quasiparticle isomer in the odd-odd nucleus 174Lu has been identified and characterized. The isomer decays to both K(pi)=7(+) and K(pi)=0(+) rotational bands obtained from the parallel and antiparallel coupling of the proton 7/2+[404] and neutron 7/2+[633] orbitals. K mixing caused by particle-rotation coupling explains the anomalously fast transition rates to the 7+ band but those to the 0+ band are caused by a chance degeneracy between the isomer and a collective state, allowing the mixing matrix element for a large K difference to be deduced.

Journal Article↗

PubMed1975

Charge properties of mitochondrial matrix proteins.

Proteins of the rat liver mitochondrial matrix have been separated into anionic (acidic), cationic(basic), and neutral groups by electrophoresis. These groups represent 69, 8, and 23% of the total matrix protein, respectively, compared to 69, 21, and 10% for the cytosol protein. The acidic nature of the mitochondrial matrix proteins has been confirmed by cellulose ion-exhange chromatography, isoelectric focusing in sucrose gradients, and amino acid analysis. The anionic, cationic, and neutral matrix proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into 18, 6, and 5 bands, respectively, compared to 22 bands for the total fraction. The significance of the charge properties of these proteins in terms of mitochondrial biogenesis is discussed.

Amino Acids↗

PubMed2004

Digital filtering implementations for the detection of broad spectral features by direct analysis of passive Fourier transform infrared interferograms.

Finite impulse response (FIR) filters and finite impulse response matrix (FIRM) filters are evaluated for use in the detection of volatile organic compounds with wide spectral bands by direct analysis of interferogram data obtained from passive Fourier transform infrared (FT-IR) measurements. Short segments of filtered interferogram points are classified by support vector machines (SVMs) to implement the automated detection of heated plumes of the target analyte, ethanol. The interferograms employed in this study were acquired with a downward-looking passive FT-IR spectrometer mounted on a fixed-wing aircraft. Classifiers are trained with data collected on the ground and subsequently used for the airborne detection. The success of the automated detection depends on the effective removal of background contributions from the interferogram segments. Removing the background signature is complicated when the analyte spectral bands are broad because there is significant overlap between the interferogram representations of the analyte and background. Methods to implement the FIR and FIRM filters while excluding background contributions are explored in this work. When properly optimized, both filtering procedures provide satisfactory classification results for the airborne data. Missed detection rates of 8% or smaller for ethanol and false positive rates of at most 0.8% are realized. The optimization of filter design parameters, the starting interferogram point for filtering, and the length of the interferogram segments used in the pattern recognition is discussed.

Journal Article↗

PubMed2002

Microplate array diagonal gel electrophoresis for cohort studies of microsatellite loci.

After PCR amplification, we have achieved precise sizing of trinucleotide and tetranucleotide microsatellite alleles on 96-well open-faced polyacrylamide microplate array diagonal gel electrophoresis (MADGE) gels: two tetranucleotide repeats, HUMTHOI (five alleles 248-263 bp) and DYS390 (eight alleles 200-228 bp), and DYS392, a trinucleotide repeat (eight alleles 210-231 bp). A gel matrix of Duracryl, a high mechanical strength polyacrylamide derivative, and appropriate ionic conditions provide the 1.3%-1.5% band resolution required. No end-labeling of primers is needed, as the sensitive Vistra Green intercalating dye is used for the visualization of bands. Co-run markers bracketing the PCR fragments ensure accurate sizing without inter-lane variability. Electrophoresis of multiple gels in a thermostatically controlled tank allows up to 1000 samples to be run in 90 min. Gel images were analyzed using a Fluorlmager 595 fluorescent scanning system, and alleles were identified using Phoretix software for band migration measurement and Microsoft Excel to compute fragment sizes. Estimated sizes were interpolated precisely to achieve accurate binning. Microsatellite-MADGE represents a utilitarian methodfor high-throughput genotyping in cohort studies, using standard laboratory equipment.

Chromosomes, Human, Y↗

PubMed2000

Purification and characterization of a feline hepatic insulin receptor.

OBJECTIVE: To elucidate the functional characteristics of a highly purified soluble liver insulin receptor in cats. SAMPLE POPULATION: Frozen livers from domestic cats were obtained commercially. PROCEDURES: The feline hepatic insulin receptor was purified from Triton X-100 solubilized plasma membranes by the use of several chromatography matrices, including affinity chromatography on an insulin-Sepharose matrix. RESULTS: The receptor, although not homogeneous, was purified 3,000-fold. Two silver-stained protein bands were identified following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with molecular weight of 134,000 and 97,000, which are similar to insulin receptors isolated from other animals. This isolated receptor had steady-state insulin binding by 40 minutes at 24 C. Optimal insulin binding occurred at pH 7.8 and with 150 mM NaCl. Under these conditions, a curvilinear Scatchard plot was obtained with the isolated receptor. Using a 2 binding-site model, the feline insulin receptor had a high-affinity low-capacity site with a dissociation constant (KD; nM) of 3 and a low-affinity high-capacity site with a K(D) of 1,180. The receptor also had tyrosine kinase activity toward an exogenous substrate that was stimulated by insulin and protamine. CONCLUSIONS AND CLINICAL RELEVANCE: Many of the reported characteristics of the liver insulin receptor in cats are similar to those for the receptor isolated from other animals and tissues, although some differences exist. These similarities suggest that characterization of the feline insulin receptor is important to understanding insulin resistance in cats with diabetes as well as in humans with diabetes.

Animals↗

PubMed2001

Localization of pigment epithelium derived factor (PEDF) in developing and adult human ocular tissues.

PURPOSE: To localize pigment epithelium-derived factor (PEDF) in developing and adult human ocular tissues. METHODS: PEDF was localized in fetal and adult eyes by immunofluorescence with a polyclonal antibody (pAb) against amino acids 327-343 of PEDF, or a monoclonal antibody (mAb) against the C-terminal 155 amino acids of PEDF. Specificity of the antibodies was documented by Western blotting. PEDF mRNA was localized in adult retina by in situ hybridization. RESULTS: In developing retinas (7.4 to 21.5 fetal weeks, Fwks), pAb anti-PEDF labeled retinal pigment epithelium (RPE) granules, developing cones, some neuroblasts and many cells in the ganglion cell layer (GCL). In adult retinas, pAb anti-PEDF labeled rod and cone cytoplasm and nuclei of rods but not cones. Cells in the INL and GCL, choroid, corneal epithelium and endothelium, and ciliary body were also pAb PEDF-positive. Preadsorption of pAb anti-PEDF with the immunizing peptide blocked specific labeling in retina and other tissues, except for photoreceptor outer segments. In agreement with the immunolocalization with pAb anti-PEDF, in situ hybridization revealed PEDF mRNA in the RPE, photoreceptors, inner nuclear layer cells and ganglion cells in adult retina. In developing retinas 18 Fwks and older, and in adult retinas, mAb anti-PEDF labeled the interphotoreceptor matrix (IPM). Western blots of retina, cornea, and ciliary body/iris with pAb anti-PEDF produced several bands at about 46 kDa. With mAb anti-PEDF, retina produced one band at about 46 kDa; cornea and ciliary body/iris had several bands at about 46 kDa. CONCLUSIONS: PEDF, originally reported as a product of RPE cells, is present in photoreceptors and inner retinal cell types in developing and adult human eyes. Photoreceptors and RPE may secrete PEDF into the IPM.

Aged↗

PubMed1992

Immunoblot analysis of antibody responses to Shigella dysenteriae type 1 infection.

Immunoblot analysis was used to identify the antigenic components of the outer membrane protein (OMP) extract from Sh. dysenteriae type 1 that may be relevant in protection against infection. The OMPs were extracted by ultrasonic disruption followed by Sarkosyl extraction. The macromolecular bands were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), electroeluted to a nitrocellulose matrix and complexed with peroxidase conjugated antibodies in human convalescent sera. IgG specific reaction was found to a major antigenic component at 57 kilodaltons. In addition, weakly reactive three antigenic determinants of greater than 57 kD and four of less than 57 kD were found. Control sera from a rabbit immunized against OMP also exhibited a similar pattern of antigenic reactivity. Some of the OMPs with high antigenicity may be important in immunoprophylaxis.

Antibodies, Bacterial↗

PubMedDate not supplied

[Optical and ESR-spectroscopic study of electronic adducts of oxymyoglobin and oxyhemoglobin].

It has been shown that low temperatures (77 degrees K) irradiation of frozen water-glycerol solutions of oxymyoglobin and oxyhemoglobin induces kinetically stabilized nonequilibrium electronic adducts (MbO2-, HbO2-) at the expense of binding of thermolyzed electrons formed during matrix radiolysis to oxygenated hem iron. The absorption spectra of HbO2-and MbO2- have a wide band with the maximum at 545 nm and Soret's band at 421 nm. At 77 K MbO2- gives the ESR spectrum with g beta 1 = 2.203 and g beta 2 = 2.103. Unlike the latter HbO2- ESR spectrum consists of two signals g beta 1 = 2.234, g beta 2 = 2.135 and g alpha 1 = 2.195, g alpha 2 = 2.103. Two signals in HbO2- spectra are shown to be conditioned by electronic adducts of oxygenated alpha- and beta-subunits. The observed effect points to non-equivalency of O2 in alpha- and beta-subunits of oxyhemoglobin. Binding of inositolhexaphopshate to oxyhemoglobin induces changes in the electron structure of HbO2-active centres.

Electron Spin Resonance Spectroscopy↗