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At least 235 records · Page 13Linked to original sources
PubMed1984

Specific photoaffinity labeling of the cAMP surface receptor in Dictyostelium discoideum.

The recent observation that ammonium sulfate stabilizes cell-surface [3H]cyclic AMP binding in Dictyostelium discoideum (Van Haastert, P., and Kien, E. (1983) J. Biol. Chem. 258, 9636-9642) led us to attempt to identify the surface cAMP receptor by photoaffinity labeling with 8-azido-[32P]cAMP using this stabilization technique. 8-azido-[32P]cAMP specifically labeled a polypeptide which migrates as a closely spaced doublet (Mr = 40,000 to 43,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Greater than 60% of the labeled polypeptide was found associated with membranes. This protein was distinguished from the cytosolic regulatory subunit of the cAMP-dependent protein kinase (Mr = 41,000) by differences in developmental regulation, specificity, and subcellular localization. No kinase regulatory subunit was detected in membranes by western blot analysis. Our preliminary observations show that labeling of this doublet correlates closely with cAMP-binding activity, suggesting that it is the surface receptor which mediates chemotaxis and cAMP signaling.

Azides↗
PubMed2005

G-matrix Fourier transform NOESY-based protocol for high-quality protein structure determination.

A protocol for high-quality structure determination based on G-matrix Fourier transform (GFT) NMR is presented. Five through-bond chemical shift correlation experiments providing 4D and 5D spectral information at high digital resolution are performed for resonance assignment. These are combined with a newly implemented (4,3)D GFT NOESY experiment which encodes information of 4D 15N/15N-, 13C(alipahtic)/15N-, and 13C(aliphatic)/13C(aliphatic)-resolved [1H,1H]-NOESY in two subspectra, each containing one component of chemical shift doublets arising from 4D --> 3D projection at omega1:Omega(1H) +/- Omega(X) [X = 15N,13C(aliphatic)]. The peaks located at the centers of the doublets are obtained from simultaneous 3D 15N/13C(aliphatic)/13C(aromatic)-resolved [1H,1H]-NOESY, wherein NOEs detected on aromatic protons are also obtained. The protocol was applied for determining a high-quality structure of the 14 kDa Northeast Structural Genomics consortium target protein, YqfB (PDB ID ). Through-bond correlation and NOESY spectra were acquired, respectively, in 16.9 and 39 h (30 h for shift doublets, 9 h for central peaks) on a 600 MHz spectrometer equipped with a cryogenic probe. The rapidly collected highly resolved 4D NOESY information allows one to assign the majority of NOEs directly from chemical shifts, which yields accurate initial structures "within" approximately 2 angstroms of the final structure. Information theoretical "QUEEN" analysis of initial distance limit constraint networks revealed that, in contrast to structure-based protocols, such NOE assignment is not biased toward identifying additional constraints that tend to be redundant with respect to the available constraint network. The protocol enables rapid NMR data collection for robust high-quality structure determination of proteins up to approximately 20-25 kDa in high-throughput.

Bacterial Proteins↗
PubMed2006

Sub-part-per-billion monitoring of nitric oxide by use of wavelength modulation spectroscopy in combination with a thermoelectrically cooled, continuous-wave quantum cascade laser.

We used a thermoelectrically cooled, continuous-wave, quantum cascade laser operating between 1847 and 1854 cm(-1) in combination with wavelength modulation spectroscopy for the detection of nitric oxide (NO) at the sub-part-per-billion by volume (ppbv) level. The laser emission overlaps the P7.5 doublet of NO centered around 1850.18 cm(-1). Using an astigmatic multiple-pass absorption cell with an optical path length of 76 m, we achieved a detection limit of 0.2 ppbv at 10 kPa, with a total acquisition time of 30 s. The corresponding minimal detectable absorption is 8.8 x 10(-9) cm(-1) Hz(-1/2).

Journal Article↗
PubMed1997

Detection of serum insulin-like growth factor binding proteins on western ligand blots by biotinylated IGF and enhanced chemiluminescence.

A novel procedure for the detection of IGF binding capacity of IGFBPs on Western ligand blots (WLB) was developed using biotinylated IGFs as probes. The biotinylated IGF-IGFBP complexes were visualized by streptavidin-horseradish peroxidase and enhanced chemiluminescence (ECL). The procedure was found to be faster and more efficient than the conventional method with iodinated IGFs. In normal human serum a predominant doublet at 38-42 kDa and five smaller bands at 35, 34, 30, 28 and 24 kDa were detected by both methods, whereas two additional bands at 26 and 16 kDa became visible with the ECL method. In pregnancy serum only one single faint band at 30 kDa could be detected by the iodinated method. In contrast, the ECL method revealed five other bands at 42, 34, 28, 26 and 16 kDa. Besides the 38-42 kDa doublet, the 30 and 16 kDa bands reacted strongly with anti-IGFBP-3 antibodies in Western immunoblotting (WIB) and therefore were related to IGFBP-3 fragments. The technical advantages of this ECL method include an extremely short exposure time to the radiographic film and a long stability of the probe. In addition, the ECL method is a non-radioactive method, making radioprotection and radioactive waste removal unnecessary.

Biotin↗
PubMed2006

Optical detection and ionization of donors in specific electronic and nuclear spin States.

We resolve the remarkably sharp bound exciton transitions of highly enriched 28Si using a single-frequency laser and photoluminescence excitation spectroscopy, as well as photocurrent spectroscopy. Well-resolved doublets in the spectrum of the 31P donor reflect the hyperfine coupling of the electronic and nuclear donor spins. The optical detection of the nuclear spin state, and selective pumping and ionization of donors in specific electronic and nuclear spin states, suggests a number of new possibilities which could be useful for the realization of silicon-based quantum computers.

Journal Article↗
PubMed2004

Reliability of comparative genomic hybridization to detect chromosome abnormalities in first polar bodies and metaphase II oocytes.

BACKGROUND: Preimplantation Genetic Diagnosis (PGD) using FISH to analyze up to nine chromosomes to discard chromosomally abnormal embryos has resulted in an increase of pregnancy rates in certain groups of patients. However, the number of chromosomes that can be analyzed is a clear limitation. We evaluate the reliability of using comparative genomic hybridization (CGH) to detect the whole set of chromosomes, as an alternative to PGD using FISH. METHODS AND RESULTS: We have analysed by CGH both, first polar bodies (1PBs) and metaphase II (MII) oocytes from 30 oocytes donated by 24 women. The aneuploidy rate was 48%. Considering two maternal age groups, a higher number of chromosome abnormalities were detected in the older group of oocytes (23% versus 75%, P < 0.02). About 33% of the 1PB-MII oocyte doublets diagnosed as aneuploid by CGH would have been misdiagnosed as normal if FISH with nine chromosome probes had been used. CONCLUSION: We demonstrate the reliability of 1PB analysis by CGH, to detect almost any chromosome abnormality in oocytes as well as unbalanced segregations of maternal translocations in a time frame compatible with regular in vitro fertilization (IVF). The selection of euploid oocytes could help to increase implantation and pregnancy rates of patients undergoing IVF treatment.

Adult↗
PubMed1994

A new variant of von Willebrand's disease (type I Padua): doublet-organized plasma von Willebrand factor oligomers in the presence of all size multimers.

Type I von Willebrand's disease (vWd) is characterized by a concomitant decrease in plasma of von Willebrand factor antigen (vWf:Ag) and vWf ristocetin cofactor activity (vWf:RCoF), associated with the presence of all-size multimers. As a rule, there is no evidence of intrinsic abnormality in vWf. We describe a family with type I vWd with an abnormal plasma vWf multimer pattern. Analysis of plasma vWf multimeric structure by short-SDS-agarose gel electrophoresis showed an abnormal banding pattern for each vWf oligomer, which was organized as a doublet instead of the normal triplet. The electrophoretic mobility of each component appeared to be normal. Multimeric analysis on a long gel showed all the bands that were detectable in normal subjects, but unlike normals the fast moving satellite stained as the major component. The platelet vWf multimer pattern was normal. The infusion of DDAVP normalized vWf:Ag, vWf:RCoF and VIII:C, but not the abnormal multimer pattern observed on both short- and long-gel electrophoresis. The return of factor VIII/vWf complex to the baseline condition was more rapid than that observed in normal subjects or classic type I vWd patients. Analysis of the subunit fragments in the patients' plasma vWf demonstrated a relatively greater proportion, compared to the normal counterpart, of a 115(140)-kD fragment, which derives from the aminoterminal region of the mature molecule; in contrast, no intact subunit was detectable. These findings indicate a new, previously unreported, variant of type I vWd, which is characterized by plasma vWf oligomers organized as doublets, instead of triplets. The reduced post-DDAVP half-life, and the abnormal subunit fragments of vWf, suggest a molecule characterized by an increased susceptibility to proteolytic degradation. As a result, the decrease in circulating vWf levels may be due to an instability of the abnormal vWf, rather than, or in addition to, a decrease in its synthesis.

Adult↗
PubMed1988

A study of androgen-resistant subjects indicates that the 6.7 pI/56 kDa protein in genital skin fibroblasts is related to the androgen receptor.

Two-dimensional gel electrophoresis of cultured human skin fibroblast lysates reveals a silver-stained "spot" of molecular mass 56 kilodaltons (kDa) and isoelectric point (pI) 6.7, occasionally as part of a doublet with a minor pI 6.5 partner. Its presence in each of 23 genital skin fibroblast strains (6 labium majus, 17 prepuce) and its absence in 30 of 32 control non-genital skin fibroblast strains accords with the 3-fold greater concentration of androgen-receptor activity in the former. However, the size and intensity of the spot do not change when cells are preincubated for 48 hours with 3 nM methyltrienolone (MT, a non-metabolizable androgen), and it is pulse-labeled with [35S]methionine to an autoradiographically equal extent, with or without incubation in 3 nM MT for 2 or 16 hours. Furthermore, the protein identified by the spot is found in the labium majus skin fibroblast strains from 2 of 12 unrelated subjects with complete androgen resistance due to negligible androgen-receptor activity, but it is absent from those of 2 others who have the same phenotype despite a normal level of qualitatively abnormal androgen-receptor activity. Hence, it is very unlikely to be an androgen-induced protein, and it cannot be a functional version of the androgen receptor itself. Its absence in 12 of 14 labium majus strains of subjects with complete androgen resistance, regardless of 5 alpha-reductase activities, indicates that it is neither a constitutive cytotypic marker of genital skin fibroblast differentiation nor a reflection of that enzyme. When intact prepuce fibroblasts are covalently labeled by photolysis with 50 nM [3H]MT, the only specific labeling detectable after two-dimensional electrophoresis is in the 6.7 and 6.5 pI doublet of the 56 kDa protein. Considering the sensitivity of silver staining and the incomplete concordance between the androgen-receptor activity of a strain and the size/intensity of its 6.7 pI/56 kDa spot on the gels, we postulate the latter to be a comparatively abundant androgen-binding protein that is causally related to the androgen receptor. The precise nature of this relation remains to be elucidated by use of novel immunologic and/or nucleic acid probes for this protein and for the mature androgen receptor. In any event, the presence or absence of the 6.7 pI/56 kDa protein in genital skin fibroblast lysates is a new marker of genetic heterogeneity within the class of complete androgen resistance.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1992

Optimal EPR detection of weak nitroxide spin adduct and ascorbyl free radical signals.

We have investigated the optimal nominal power settings for the electron paramagnetic resonance detection of typical nitroxides, nitroxide spin adducts, and the ascorbyl free radical. In room temperature aqueous solution, we find that, for all the nitroxides examined, saturation effects begin at approx. 25 mW nominal power with maximum signal intensity achieved at approx. 100 mW power when using a TM110 cavity. For the ascorbyl free radical, we find that saturation effects begin at approx. 16 mW nominal power and that maximum peak-to-peak signal amplitude is obtained at approx. 40 mW microwave power. For the ascorbyl free radical, we find that a modulation amplitude of approx. 0.65 G yields the maximum signal height for the doublet signal. This information will help researchers maximize the EPR signal height of minimally detectable free radicals such as encountered in biological systems.

Ascorbic Acid↗
PubMed1997

Dimethylsulfoxide maintains intercellular communication by preserving the gap junctional protein connexin32 in primary cultured hepatocyte doublets from rats.

Intercellular communication via gap junctions is one of the differentiated functions of cells. Dimethylsulfoxide (DMSO) is known to induce cell differentiation and maintain differentiated cellular functions in primary hepatocyte culture, but the mechanism of action of DMSO is unknown. Therefore, we investigated the effect of DMSO on cell-cell communication via gap junctions of hepatocyte doublets, which are differentiated cells that lose differentiated functions with time in culture. In isolated rat hepatocyte doublets, we assessed the effects of 1, 2 and 3% DMSO in culture medium on morphological changes and dye-coupling activity between pairs of cells by microinjection with fluorescent dye (Lucifer Yellow CH). The distribution of gap junction protein connexin32 (Cx32) was assessed by indirect immunofluorescence analysis and the Cx32 mRNA was detected by the reverse transcription-polymerase chain reaction method. Dimethylsulfoxide delayed the morphological change of hepatocyte doublets from a spherical to a flattened shape. Dye-coupling efficiency significantly decreased with time in culture in the control group, whereas in groups treated with 2 and 3% DMSO, dye-coupling efficiency was retained after 6 and 9 h of inoculation (P < 0.05 and P < 0.01, respectively). Analysis by indirect immunofluorescence showed few fluorescent spots for Cx32 in the control group at 9 h of incubation, whereas many punctate fluorescent spots were seen in the 3% DMSO group at 9 h of incubation. The detection of Cx32 mRNA in the 3% DMSO group was also stronger than in controls. Dimethylsulfoxide significantly maintained intercellular communication via gap junctions in primary cultured rat hepatocytes through the preservation of functional Cx32 protein, thus maintaining cell differentiation.

Animals↗
PubMed2001

Circulating tumor cell clusters in the peripheral blood of colorectal cancer patients.

PURPOSE: Recently several reverse transcription-PCR techniques have been proven to be useful for the detection of circulating micrometastases. However, this way intact cell clusters that were found in animal experiments of prognostic value could not be detected. In this study, evaluation and modification of a commercial, cytokeratin-based, immunomagnetic cell separation method was performed for the detection of intact cell clusters in colorectal carcinoma patients. EXPERIMENTAL DESIGN: Thirty-two colon cancer patients (6 were in Dukes stage B, 13 in stage C, and 13 in stage D) and 20 healthy donor samples were evaluated. Immunomagnetic cell separation was performed from the buffy coat of peripheral blood samples (20 ml) using the Carcinoma Cell Enrichment Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), avoiding any filtering steps. The enriched cell fraction was cytocentrifuged and immunocytochemically labeled using a pancytokeratin antibody (MNF116; Dako). RESULTS: Of 20 healthy samples, 2 contained one cytokeratin-positive cell. Of 32 single samples from malignant cases, 24 showed cytokeratin-positive cells. Tumor cell clusters, mixed-cell doublets (one cytokeratin-positive and -negative cell), and mixed-cell clusters were detected in 22 of 24 patients. In six cases, cytokeratin-positive dendritic-like cells were detected. Follow-up data indicate that chemotherapy cannot destroy all of the circulating tumor cell clusters. CONCLUSIONS: Using the methods presented, we could detect circulating colon cancer cells and cell clusters in colon carcinoma patients. Similar cellular structures were described previously only in rats. Present data prove that such structures are present in human colorectal cancer, too.

Cell Adhesion↗
PubMed1994

Electron micrograph map of the Drosophila melanogaster polytene chromosome 3R divisions 81 through 90.

The banding pattern of the proximal half of the polytene salivary gland 3R chromosome of Drosophila melanogaster was studied with thin section electron microscopy. Bands were identified according to Bridges' revised light microscopic map, which contains 330 single and 121 double bands within the regions 81 through 90. We found a total of 447 bands in this region. 97 Bridges' single bands were easily detected in almost all thin sections, while 177 faint bands could be seen only in some micrographs. 56 Bridges' single bands could not be found. 32 Bridges' doublets were made up of two separate bands each in thin sections. The other 89 Bridges' doublets seemed to be either single bands or remained obscure. A total of 20 small new bands, which were not drawn on Bridges' map, were detected.

Animals↗
PubMed1990

Temporal correlations in modulated evoked responses in the visual cortical cells of the cat.

We analysed evoked responses recorded from 97 cells in the visual cortex of 4 adult cats and 8 kittens, stimulated by a drifting sinusoidal grating. A Fourier analysis of the responses allowed us to select 30 cells showing a clear modulating response (relative modulation index greater than 1). The 162 records from these selected cells were scanned to detect precise temporal correlations in the form of replicating triplets and associated "ghost" doublets. Temporal correlations of this nature were observed in these cells. They are about 10 times more abundant in adult cats than in kittens, and mostly observed in infragranular cortical layer cells. The possible role of these precise temporal patterns in information processing in the brain is examined, as well as the relation between this type of temporal correlation with coherent oscillations and principal components waveforms.

Animals↗
PubMed1998

A Plasmodium chabaudi protein contains a repetitive region with a predicted spectrin-like structure.

cDNA and genomic DNA clones covering the entire open reading frame (ORF) for a Plasmodium chabaudi 96V protein were isolated. From the first ATG codon the intronless gene codes for a 229-kDa protein. Antisera raised against recombinant polypeptides coded by two different regions of the gene reacted with a 240/225-kDa doublet on Western blots of parasite extracts. In immunofluorescence studies the same sera detected the antigen at the apical end of the merozoite, possibly in rhoptry organelles. In Western blotting experiments the recombinant polypeptides were recognised by antibodies induced by natural infection. A 364-amino acid residue repetitive region, based on 32 11-mer repeats divided by two 6-mer repeats into three blocks, is located in the centre of the protein. Analysis of this repetitive region led us to propose a model in which each of the three units forms an alpha-helical coiled-coil triple-helix containing a possible leucine-histidine zipper. Each unit resembles in structure the units present in spectrin. The repeat region is flanked by predicted heptad based alpha-helical coiled-coil regions, and we propose that the protein forms a dimer. The 229-kDa protein has the overall character of a cytoskeletal protein. We have named the 229-kDa protein repetitive organellar protein (ROPE) and suggest that ROPE may be involved in the process of invasion, possibly by interacting with the erythrocyte cytoskeleton, and that the leucine histidine-zipper may be involved in molecular mimicry of spectrin.

Amino Acid Sequence↗
PubMed1977

Identification of dynein as the outer arms of sea urchin sperm axonemes.

The location of dynein, the main flagellar ATPase, within the sea urchin sperm axoneme was investigated by the use of immunofluorescence and immunoelectron microscopy, employing an antiserum against a tryptic fragment of dynein 1 (Fragment 1A) purified from sea urchin sperm flagella. The axonemes were found to be stained with the antiserum when examined by an indirect immunofluorescence technique. Immunoelectron microscopy with the antiserum and a ferritin-conjugated IgG fraction of goat antiserum to rabbit IgG revealed that, among the structures within the axoneme, only the outer arms were labeled with ferritin particles. With either the normal serum or antiserum absorbed with Fragment 1A, there were no ferritin particles within the axonemes. When the outer arms were extracted with 0.5 M NaCl, leaving the inner arms intact, again no ferritin dots were detected. Furthermore, it was found that the outer arm on the no. 5 doublet microtubule, which connects with the extra arm projection backward from the no. 6 doublet, had no attached ferritin particles. From these observations, it can be concluded that the outer arm consists of dynein (at least dynein 1) and that Fragment 1A, containing the active site for ATPase activity of dynein 1, is located at the distal end of the outer arms. The significance of the present findings is considered in connection with flagellar movement.

Adenosine Triphosphatases↗
PubMed1986

A microtubule-activated ATPase from sea urchin eggs, distinct from cytoplasmic dynein and kinesin.

We report an ATPase activity, present in sea urchin egg cytosol, that is activated by microtubules. The activity sediments at 10 S in sucrose gradients and is clearly distinct from activities at 12 S and 20 S due to cytoplasmic dynein. Potent activation of the ATPase is observed when endogenous egg tubulin is induced to assemble with taxol or when exogenous taxol-stabilized pure brain tubulin microtubules or flagellar outer-doublet microtubules are added. No activation by tubulin subunits or taxol alone is detectable. In contrast to flagellar or cytoplasmic dynein, the microtubule-activated enzyme is unaffected by vanadate or by nonionic detergents and hydrolyzes GTP in addition to ATP. In contrast to kinesin, it cosediments with microtubules in the presence or absence of ATP. The microtubule-activated enzyme may have a role in microtubule-based motility.

Adenosine Triphosphatases↗
PubMed1993

Transfection of the human insulin-like growth factor binding protein-3 gene into Balb/c fibroblasts inhibits cellular growth.

The insulin-like growth factors (IGFs) are potent mitogens with both endocrine and autocrine-paracrine effects on cell growth. The IGF binding proteins (IGFBPs) are found in many human biological fluids and in the conditioned media of many cell cultures. These molecules are ontogenically and hormonally regulated. Nevertheless, the biological role(s) of the IGFBPs remain controversial. Both inhibitory and stimulatory effects of IGFBPs on cell growth have been suggested. In order to evaluate the actions that endogenously produced IGFBPs have on cell growth, we constructed a mammalian expression vector containing the human IGFBP-3 cDNA and transfected the murine Balb/c cell line. While plasmid-transfected control Balb/c cells (Tx-P) produced only small amounts of a 28-kilodalton IGFBP, the IGFBP-3-transfected Balb/c cells (Tx-BP-3) additionally expressed readily detectable amounts of the characteristic 40- to 44-kilodalton human IGFBP-3 protein doublet and its mRNA. Growth of Tx-BP-3 in serum-containing media was significantly (2.5-fold) slower compared with Tx-P grown under identical conditions. Fully confluent Tx-BP-3 cells arrested their growth at a cell density that was 3-fold lower than did Tx-P cells. When nontransfected cells were grown in the presence of high concentrations of either IGF-I or insulin, growth was equally stimulated. However, when transfected cells were grown in insulin-containing media (5 micrograms/ml), growth rates of the IGFBP-3-transfected cells were not restored to those observed in plasmid-transfected control cells. These results suggest that in this model, the expression of endogenous IGFBP-3 has an inhibitory effect on cell growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Aberrant processing of alkaline phosphatase precursor caused by blocking the synthesis of glycosylphosphatidylinositol.

Alkaline phosphatase is anchored to the membrane via glycosylphosphatidylinositol (GPI). Mannose residues of the GPI glycan are suggested to be derived from dolichol-P-mannose. In the present study we examined the effect of 2-fluoro-2-deoxy-D-glucose (F-Glc), an inhibitor of dolichol-P-mannose synthesis, on the biosynthesis and processing of alkaline phosphatase in JEG-3 cells. In control cells, a proform precursor (64.5 kDa) with a hydrophobic peptide domain at the COOH terminus was immediately processed into an intermediate form (63 kDa) by proteolytic removal of the COOH-terminal extension and replacement with the GPI anchor, and then to a mature form (66 kDa) by terminal glycosylation of its N-linked oligosaccharides. In contrast, when cells were treated with F-Glc (1 mM), the protein was synthesized as a proform of 61 kDa. The reduction in its molecular mass was mostly due to the inhibition in maturation of N-linked oligosaccharides by F-Glc. The 61-kDa proform identified by antibodies to the COOH-terminal peptide was detectable even at 3 h after the synthesis, and was gradually processed to doublet forms of 58-59 kDa which were finally secreted into the medium. None of these forms were labeled with [3H]ethanolamine and [3H]stearic acid, components of the GPI anchor, and expressed on the cell surface as a membrane-bound form. Taken together, these results suggest that the inhibition of the GPI synthesis causes a prolonged accumulation of the proform, which is then gradually processed into secretory forms by proteolytic removal of the COOH-terminal hydrophobic peptide.

Alkaline Phosphatase↗