Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “doublet detection”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 307 records · Page 17Linked to original sources
PubMed2001

Chromosomal translocation 3;22 in an infertile man.

OBJECTIVE: To present the first case of an infertile male with a normal phenotype and chromosomal translocation 3;22. DESIGN: Case report. SETTING: POVISA Medical Center. PATIENT(S): A 45-year-old man with primary infertility for 13 years and with different partners; the patient has a family history of recurrent miscarriages and low fertility. INTERVENTION(S): Lymphocytic karyotype and electron microscopy. MAIN OUTCOME MEASURE(S): Physical examination and semen analysis. RESULT(S): The semen analysis revealed oligoasthenoteratospermia. The lymphocytic karyotype detected a translocation 3;22, and electron microscopy showed a lack of the central microtubule pair and peripheral doublet. CONCLUSION(S): An association between translocation 3;22 and other abnormalities in infertile males has been reported, but no such association has ever been described in men whose only clinical manifestation is infertility.

Chromosomes↗
PubMed1998

Identification of a specific effector of the small GTP-binding protein Rap2.

Rap2 is a small GTP-binding protein that belongs to the Ras superfamily and whose function is still unknown. To elucidate Rap2 function, we searched for potential effectors by screening a mouse brain cDNA library in a yeast two-hybrid system using as a bait a Rap2A protein bearing a mutation of Gly to Val at position 12. This strategy lead to the identification of a protein that interacts specifically with Rap2A complexed with GTP, and requires an intact effector domain of Rap2A for interaction; we designated this protein Rap2-interacting protein 8 (RPIP8). Biochemical data obtained from in vitro studies with purified proteins confirmed the genetic results. Mouse RPIP8 consists of 446 amino acids, bears a coiled-coil domain between residues 265 and 313, and is expressed principally in brain. Its human counterpart, of 400 amino acids, exhibits 93.7% identity in their common region. A search for similar sequences in expressed-sequence-tags databanks revealed the presence in human and rodents of mRNAs encoding the 400-residue and 446-residue forms of RPIP8. Furthermore a doublet of 45-50 kDa, corresponding to the 400-residue and 446-residue forms of the protein, was detected by western blotting of mouse brain extracts and lysates from pheochromocytoma PC12 cells and the pancreatic beta-cell lines HIT-T15 and RIN-m5F. Using transient transfections of HIT-T15 cells it was possible to demonstrate that [Val12]Rap2 and wild-type Rap2 could be immunoprecipitated with RPIP8. These data therefore argue for RPIP8 being a specific effector of the Rap2 protein in cells exhibiting neuronal properties.

Amino Acid Sequence↗
PubMed1997

gRICH68 and gRICH70 are 2',3'-cyclic-nucleotide 3'-phosphodiesterases induced during goldfish optic nerve regeneration.

Biochemical characterization of changes in gene expression that accompany optic nerve regeneration has led to the identification of proteins that may play key roles in the regeneration process. In this report, a cDNA encoding gRICH70, a novel isoform of the regeneration-induced gRICH68 protein, has been identified and characterized in goldfish. Both gRICH68 and gRICH70 show significant homology (34-36%) to mammalian 2',3'-cyclic-nucleotide 3'-phosphodiesterases (CNPases), hence the name goldfish regeneration-induced CNPase homolog (gRICH). The predicted 431-amino acid gRICH70 protein is 88% homologous to gRICH68, and the retinal mRNA for gRICH70 is coordinately induced with gRICH68 mRNA during optic nerve regeneration. Enzymatic analysis of recombinant proteins confirms that both gRICH proteins possess CNPase activity. Despite the relatively limited sequence homology, the kinetic constants obtained suggest that both gRICH proteins are at least as efficient as recombinant mouse CNP1 in catalyzing the hydrolysis of 2',3'-cAMP. Immunoprecipitation studies indicate that gRICH proteins are responsible for the majority of the CNPase activity detected in regenerating goldfish retinas. The evidence presented demonstrates that gRICH68 and gRICH70 correspond to a previously described doublet of acidic proteins that are selectively induced in the goldfish retina during optic nerve regeneration. Thus, CNPase enzyme activity is implicated for the first time in the process of nerve regeneration.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1992

Tubulin protofilaments and kinesin-dependent motility.

Microtubules are built of tubulin subunits assembled into hollow cylinders which consist of parallel protofilaments. Thus, motor molecules interacting with a microtubule could do so either with one or several tubulin subunits. This makes it difficult to determine the structural requirements for the interaction. One way to approach the problem is to alter the surface lattice. This can be done in several ways. Proto-filaments can be exposed on their inside (C-tubules or "sheets"), they can be made antiparallel (zinc sheets), or they can be rolled up (duplex tubules). We have exploited this polymorphism to study how the motor protein kinesin attached to a glass surface interacts and moves the various tubulin assemblies. Microtubules glide over the surface along straight paths and with uniform velocities. In the case of C-tubules, approximately 40% glide similarly to microtubules, but a major fraction do not glide at all. This indicates (a) that a full cylindrical closure is not necessary for movement, and (b) that the inside surface of microtubules does not support gliding. With zinc sheets, up to 70% of the polymers move, but the movement is discontinuous, has a reduced speed, and follows along a curved path. Since zinc sheets have protofilaments alternating in orientation and polarity, this result suggests that in principle a single protofilament can produce movement, even when its neighbors cannot. Duplex microtubules do not move because they are covered with protofilaments coiled inside out, thus preventing the interaction with kinesin. The data can be explained by assuming that the outside of one protofilament represents the minimal track for kinesin, but smooth gliding requires several parallel protofilaments. Finally, we followed the motion of kinesin-coated microbeads on sea-urchin sperm flagella, from the flagellar outer doublet microtubules to the singlet microtubule tips extending from the A-tubules. No change in behavior was detected during the transition. This indicates that even if these microtubules differ in surface lattice, this does not affect the motility.

Animals↗
PubMed1976

Topography of the synaptosomal membrane.

The composition and disposition of the constituent polypeptides of rat cerebral cortical synaptosomal membranes were analyzed on SDS acrylamide gels. Of 20 bands readily detected, 11 account for greater than 93% of the total protein analyzed. These are: (molecu25); 3 (175); 4 (doublet, 137); 5 (doublet, 97); 6 (68); 7 (61); 8 (54); 9 (44); 10 (37); and 11 (33). Bands 5 and 8-10 are the most prominent and account for greater than 60% of the protein mass or 0.67 of its molecular fraction. By lactoperoxidase iodination, the bulk of the proteins in bands 3, 5, 6, and 8 and a portion of band 11 appear to be located on the external (junctional) face of the membrane of intact synaptosomes; proteins in bands 1, 2, 7, 9, and 10 appear to be localized on the internal (synaptoplasmic) face and become labeled only when synaptosomes are lysed. Further confirmation of the topographical distribution is provided by evidence that bands 3-6, 8, and 11 contain glycoproteins susceptible to labeling in intact synaptosomes by oxidation with galactose oxidase or periodate followed by reduction with NaB3H4. Evidence is provided for significant contributions by tubulin- and actin-like molecules to bands 8 and 9, respectively, suggesting that a substantial fraction of the tubulin in the synaptosomal membrane is disposed externally (accessible to iodination) whereas most, if not all, of the actin appears to exhibit the opposite topography. Similar though weaker inferences can also be drawn with regard to the location of tropomyosin and troponin. Preliminary evidence is provided that postsynaptic densities exhibit a protein and iodination profile distinct from that of the synpatosomal membrane.

Actins↗
PubMed1994

Production, purification and characterization of recombinant yeast processing alpha 1,2-mannosidase.

The Saccharomyces cerevisiae processing alpha 1,2-mannosidase, which trims Man9GlcNAc to Man8GlcNAc, has a lumenally oriented catalytic domain and an N-terminal transmembrane domain. To obtain sufficient protein to study the structure and mechanism of action of this enzyme, the sequence encoding the catalytic domain was inserted downstream of the alpha-factor promoter and signal peptide in a high-copy vector for expression in S. cerevisiae as a secreted protein. Using oligosaccharide substrate (Glc1Man9GlcNAc or Man9GlcNAc), the medium of cells transformed with this plasmid showed an increase in alpha-mannosidase activity that was directly related to the increase in cell density, whereas no alpha-mannosidase activity was detected in cells transformed with vector alone. SDS-PAGE of the medium showed the presence of a doublet of 63 and 60 kDa that was revealed by Coomassie Blue staining and by Western blotting with antibodies to the endogenous solubilized alpha-mannosidase. The recombinant alpha-mannosidase was present in the medium at a level of approximately 1 mg/l and was purified in a single step by chromatography on S-Sepharose. High-resolution 1H NMR analysis of the Man8GlcNAc formed from Man9GlcNAc in the presence of the recombinant enzyme proved that it retained its specificity and removed only one specific alpha 1,2-mannose residue of the alpha 1,3 branch. Endoglycosidase H treatment decreased the molecular mass of both components of the doublet by approximately 5 kDa, showing that the heterogeneity is not due to differential N-glycosylation. EDTA inhibited the activity of the recombinant enzyme, but the inhibition was reversed by the addition of divalent cations.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Activity of cefepime and other beta-lactam antibiotics against permeability mutants of Escherichia coli and Klebsiella pneumoniae.

Permeability mutants were obtained, under selection pressure with cefoxitin, from five strains each of Escherichia coli and Klebsiella pneumoniae. Most mutants showed alterations to their outer membrane protein profiles. In general, the porins OmpC and F were lost from E. coli mutants, and one or both components of a 40 kD doublet were lost from K. pneumoniae mutants. Additional minor changes to outer membrane proteins, of unknown significance, were detected in many of the mutants. MICs of cefepime, ceftazidime and cefpirome for the porin-deficient mutants were up to 128-fold above those for their parent strains, but remained < or = 4 mg/L, whereas those of cefoxitin, cefuroxime, and penicillins were raised above accepted breakpoints. The detection of reduced susceptibility to third- and fourth-generation cephalosporins is in contrast to the findings of other groups. Most (9/10) mutants with high-level cefoxitin resistance were reasonably stable during sequential batch culture in rich media, but susceptible variants tended to overgrow under nutrient depletion in continuous culture. These variants were not true revertants, having failed to regain the major porins: rather they appeared to have undergone compensatory mutations. Their instability probably explains why porin-deficient enterobacteria have not become a significant clinical problem, despite the ease with which they can be selected in vitro.

Anti-Bacterial Agents↗
PubMed1987

Polymorphic keratins in human epidermis.

Human epidermal keratins from many different individuals were identified and compared by both high-resolution 1- and 2-dimensional gel electrophoresis and immunoblotting. While the polypeptide patterns obtained for keratin-enriched cytoskeletal preparations could be considered typical of normal interfollicular epidermis, they also disclosed variations, among the individuals, concerning some of the constituent protein subunits. Three sets of interindividually varying keratins could be distinguished owing to their distinct, though small, differences in electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels and their similar or identical charge characteristics upon nonequilibrium pH gradient electrophoresis: the basic keratins 1a and 1b as well as 5a and 5b and the acidic keratins 10a and 10b. Of each set either a doublet, showing a marked 1:1 ratio of polypeptides, or the one or the other variant protein was detected together with keratin 14, which did not display any variation in a series of 148 individual tissue samples tested. Thus, the keratin composition of human epidermis could be summarized in the formula: (1a v 1b) + (5a v 5b) + (10a v 10b) + 14. The systematic appearance of the variants suggested that each protein within a set is the product of an independent allele. In support of this hypothesis we have found that the same variant is expressed in other epithelia of a given individual. Moreover, the frequency of any of the keratins in our sampling concurred with the frequency predicted by the Hardy-Weinberg relation for the distribution of alleles in a population, as did the frequency distribution of particular keratin patterns.

Alleles↗
PubMed1992

20-Hz pulses and other vocalizations of fin whales, Balaenoptera physalus, in the Gulf of California, Mexico.

Low-frequency vocalizations were recorded from fin whales, Balaenoptera physalus, in the Gulf of California, Mexico, during three cruises. In March 1985, recorded 20-Hz pulses were in sequences of regular 9-s interpulse intervals. In August 1987, nearly all were in sequences of doublets with alternating 5- and 18-s interpulse intervals. No 20-Hz pulse sequences of any kind were detected in February 1987. The typical pulse modulated from 42 to 20 Hz and its median duration was 0.7 s (1985 data). Most other fin whale sounds were also short tonal pulses averaging 82, 56, and 68 Hz, respectively, for the three cruises; 89% were modulated in frequency, mostly downward. Compared to Atlantic and Pacific Ocean regions, Gulf of California 20-Hz pulses were unique in terms of frequency modulation, interpulse sound levels, and temporal patterns. Fin whales in the Gulf may represent a regional stock revealed by their sound characteristics, a phenomenon previously shown for humpback whales, birds, and fish. Regional differences in fin whale sounds were found in comparisons of Atlantic and Pacific locations.

Animal Communication↗
PubMed1996

Tumor necrosis factor alpha stimulates insulin-like growth factor binding protein 3 expression in cultured porcine Sertoli cells.

The present study was undertaken to analyze how a cytokine, the tumor necrosis factor alpha (TNF alpha), antagonizes the stimulatory action of insulin-like growth factor-I (IGF-I) on FSH receptor levels in testicular Sertoli cells. To achieve this purpose, we measured, in a model of primary culture of porcine Sertoli cells, the effects of TNF alpha on the IGF system that includes IGF-I and IGF-II, IGF binding proteins (IGFBPs), and IGF-I receptor (IGF-I R). We report that while TNF alpha had no consistent effect on the levels of IGF-I, IGF-II, or on IGF-I receptor (protein and messenger RNA (mRNA)], it stimulated IGFBP activity and particularly IGFBP-3. TNF alpha stimulated predominantly IGFBP-3 (about 4-fold) both in terms of mRNA (a 2.6-kilobase transcript, measured by Northern blotting analysis), and protein (a doublet of 40-44 kDa, assessed by ligand blotting analysis). Such a stimulatory effect on IGFBP-3 was detected with a concentration as low as 0.1 ng/ml (5.5 pM) of TNF alpha. The stimulatory action of the cytokine was time dependent and was maximal at 7 h and 48 h, for IGFBP-3 mRNA and protein, respectively. Such an increase in IGFBP-3 in TNF alpha-treated Sertoli cells results probably in a decrease in IGF-1 bioavailability for its receptors and thus in a decrease in IGF-I action. Indeed, addition of recombinant human IGFBP-3 (10 nM) suppressed completely the stimulatory addition of IGF-I (3 nM) on FSH binding to cultured porcine Sertoli cells. Together, the present findings indicate that, in Sertoli cells, TNF alpha antagonizes IGF-I action through the modulation of IGFBPs and particularly through an increase in IGFBP-3. Because of the local production of both TNF alpha and components of the IGF system, such an interaction between the IGF system and the cytokine probably occurs in the context of physiological testicular somatic-germ cell interactions.

Animals↗
PubMed1993

Expression of the protein product of the prostaglandin synthase-2/TIS10 gene in mitogen-stimulated Swiss 3T3 cells.

TIS10/PGS-2 encodes a prostaglandin synthase (PGS) distinct from the previously described enzyme PGS-1 (EC 1.14.99.1). We have now generated antipeptide antisera, directed to an amino acid sequence unique to the murine TIS10/PGS-2 protein, which specifically recognize the TIS10/PGS-2 antigen. TIS10/PGS-2 protein was undetectable in quiescent Swiss 3T3 cells. The level of TIS10/PGS-2 protein peaked between 6 and 8 h following phorbol ester stimulation of cells, then declined to basal levels after 18-24 h. Synthesis of TIS10/PGS-2 protein was dramatically increased in the second hour following mitogen stimulation and remained elevated for several hours. The half-life of the TIS10/PGS-2 protein was 4 h. Immunofluorescence studies demonstrated a perinuclear and cytoplasmic localization of the TIS10/PGS-2 antigen. As expected, detection of induced TIS10/PGS-2 antigen was dependent on protein synthesis. Metabolically labeled TIS10/PGS-2 protein migrated as a 71/73-kDa doublet following immunoprecipitation. Dexamethasone blocked both the TPA- and serum-induced appearance of TIS10/PGS-2 antigen. These studies demonstrate the existence of a mitogen-inducible, glucocorticoid-inhibitable, immunologically distinct prostaglandin synthase protein.

3T3 Cells↗
PubMed2000

Irregular costameres represent nitric oxide synthase-1-positive sarcolemma invaginations enriched in contracted skeletal muscle fibres.

NADPH diaphorase histochemistry and NOS-1 immunohistochemistry on 60 microm thick frozen sections of rat extensor digitorum longus muscles led to the detection of prominent rings clearly encompassing the surface of the muscle fibres. These so far unknown costameres were usually found as doublets flanking a space of about 2 microm width. Because these costameric doublets did not appear in regular periods, we designate them irregular costameres to discriminate them from regular ones with a 1 microm periodicity overlying Z-discs and M-lines. Irregular costameres were thicker than the regular ones and free of intercostameres. Immunohistochemistry demonstrated that NOS-1 was co-localized with integral (beta-dystroglycan, alpha-sarcoglycan) and peripheral (caveolin-3, dystrophin) members of the enlarged dystrophin complex in the irregular costameres but not with non-sarcolemmal organized proteins (myosin heavy chain, alpha-actinin, desmin and sarcoplasmic reticulum-located Ca2+-dependent ATPase-1). Invaginations of the sarcolemma to form irregular costameres were observed. In teased myofibres the sarcolemma between two following irregular costameres was ballooned, while the irregular costameres themselves clamped the fibres together. Finally, the number of detectable irregular costameres was significantly increased in maximally contracted extensor digitorum longus muscles generated by electric stimulation but decreased in mechanically stretched ones. Combining these observations, we hypothesize that irregular costameres belong to a reserve zone for the sarcolemma necessary for the contraction/relaxation cycle in myofibres.

Animals↗
PubMed2003

Connective tissue growth factor and its regulation in the peritoneal cavity of peritoneal dialysis patients.

BACKGROUND: Connective tissue growth factor (CTGF) is a fibrogenic cytokine that is highly expressed in wound healing and fibrotic lesions. The role of transforming growth factor-beta (TGF-beta) in fibrosis is well documented, and the emerging understanding that its fibrogenic actions are mediated through CTGF has provided an attractive target molecule for the modulation of matrix overproduction in fibrotic disease. The involvement of CTGF in the pathogenesis of peritoneal membrane fibrosis in peritoneal dialysis (PD) patients has not been investigated, and so the aim of this study was to ascertain whether CTGF is produced in the peritoneal cavity of PD patients and to investigate its regulation by cytokines. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR), Northern blotting, and Western blotting were used to study CTGF expression by cultured human peritoneal mesothelial cells (HPMC) from peritoneal dialysis patients. Western blotting was used to detect CTGF expression in spent peritoneal dialysate from patients with and without peritonitis. RESULTS: RT-PCR analysis demonstrated the expression of CTGF mRNA in cultured primay HPMCs isolated from spent peritoneal effluent. The production of the major 36 to 38 kD CTGF protein doublet by HPMC in addition to a 23 to 25 kD proteolytically processed form was confirmed by Western blotting. Several molecular weight forms of CTGF (18 to 38 kD) were also detected by Western blotting in peritoneal dialysate, with levels markedly elevated during episodes of peritonitis. Northern and Western blot analysis revealed that CTGF mRNA and protein production by HPMC was up-regulated by TGF-beta, with mRNA levels significantly increasing above the control (P < 0.01). In contrast, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and tumor necrosis factor-alpha (TNF-alpha) had no measurable effects on CTGF mRNA expression. CONCLUSION: These results are the first to demonstrate the production of CTGF by HPMC and its presence in the peritoneal cavity of PD patients. The marked increase in CTGF levels by factors implicated in the development of peritoneal membrane fibrosis suggests its involvement in the underlying pathophysiologic mechanism(s).

Base Sequence↗
PubMed1997

Identification of a novel protein encoded by the BamHI A region of the Epstein-Barr virus.

An unusual set of 3' coterminal, spliced mRNAs transcribed through the BamHI A fragment have been previously identified in nasopharyngeal carcinoma (NPC) tissues. These RNAs have also been detected at low levels in Burkitt's lymphoma (BL) cell lines and Epstein-Barr virus (EBV)-transformed lymphocytes. Sequence analyses of clones from a cDNA library derived from the C15 NPC tumor indicated that the primary transcripts are differentially spliced, giving rise to a family of related transcripts, all of which encompass the BARF0 open reading frame (ORF) at the 3' end of the transcripts. One cDNA was identified that extended the BARF0 ORF at the 5' end, forming the RK-BARF0 ORF. In this study, a rabbit antiserum to a synthetic peptide representing an amino acid sequence encoded by the BARF0 ORF was prepared. This antiserum detected a glutathione S-transferase-BARF0 fusion protein and both BARF0 and RK-BARF0 proteins expressed from transfected constructs in H1299 cells. The serum also immunoprecipitated the 20-kDa BARF0 and 30-kDa RK-BARF0 in vitro-translated proteins. Immunoblot analyses identified a protein doublet of 30 and 35 kDa in all of the EBV-infected cell lines tested. Cellular fractionation studies revealed that the proteins were membrane associated. The sizes of the proteins detected in cell lines and their association with membranes suggests that they are likely encoded by the RK-BARF0 transcript, which is predicted to contain a membrane localization signal. The proteins were also detected in protein extracts prepared from NPC biopsies and a BL biopsy but not from hairy leukoplakia, a permissive EBV infection. These results reveal that the rightward RNA transcripts from the BamHI A region of EBV encode one or more proteins that are expressed in latently infected cells and in tumor tissue.

Amino Acid Sequence↗
PubMed1991

Motion-insensitive volume-selective pulse sequences for direct and proton-detected 13C spectroscopy: detection of glycogen in the human liver in vivo.

Two compact pulse sequences are reported for the volume-selective detection of 13C nuclei. 13C signals are either directly acquired after enhancing the amplitude by polarization transfer from the coupled protons or, preferably, indirectly detected by heteronuclear editing of proton signals of 1H nuclei coupled to 13C. In the latter case, the full sensitivity of proton resonance is achieved, and signals from uncoupled protons or protons coupled to non-13C nuclei or 13C nuclei of undesired compounds are suppressed. Both sequences are single-scan procedures and are insensitive to the pulse phases and to motions of the investigated organs. The insensitivity to organ motions is due to the extremely compact character of these sequences avoiding coherence evolution periods as far as possible. This is achieved first by the introduction of double-resonance sandwich (DORSA) pulses accomplishing the polarization transfer slice selectively in a very short time. The second reason is that the initial evolution interval which is usually part of polarization transfer experiments is avoided by the aid of a doublet line-selective inversion pulse. Several test experiments in vivo are reported. In particular, it is demonstrated that glycogen can be detected in natural abundance in the human liver in vivo using a 2-T whole-body tomograph even without the use of heart or respiration triggers. The total acquisition time was 22 min, and a signal-to-noise ratio of 6 was achieved.

Carbon↗
PubMed1986

Cytoskeletons of ADP- and thrombin-stimulated blood platelets. Presence of a caldesmon-like protein, alpha-actinin and gelsolin at different steps of the stimulation.

Comparative analyses of the cytoskeletons of resting and stimulated platelets point out the involvement of a 79 kDa polypeptide in the activation step and its increased incorporation during aggregation. It appears as a doublet and cross-reacts with an antibody to chicken gizzard caldesmon, whereas no 150 kDa immunoreactive form was detected. alpha-Actinin and gelsolin were detected only in the aggregation step.

Actinin↗
PubMed1995

Studies on the role of glycosylation in the origin of the electrophoretic variants for rat corticosteroid-binding globulin.

The glycoprotein corticosteroid-binding globulin (CBG) migrates as doublet bands in PAGE and SDS-PAGE, and as numerous bands in isoelectric focusing (IEF). This study deals with the origin of this heterogeneity. Desialation of rat CBG with neuraminidase does not abolish the doublet in either PAGE or SDS-PAGE, indicating that the doublet does not arise as a result of differences in sialic acid residues. Treatment of the separated upper and lower variants of native CBG with N-glycosidase F (PNGase-F) shows a differential pattern of deglycosylation over time indicating either differences in the number, type, or location of sugars attached to each of the variants. Rate of deglycosylation is quicker and more extensive for the upper variant when compared to the lower variant. PNGase-F treatment of 1% SDS-denatured CBG does not abolish the CBG doublet seen in SDS-PAGE, indicating that there is variation in the protein moiety. Sugars could not be detected on PNGase-F treated CBG using either wheat germ aglutinin horse radish peroxidase conjugate, concavilin-A HRP conjugate, or the digoxigenin glycan detection system. While the results clearly show differences in glycosylation between the CBG variants, differences in the protein moiety may also occur to give rise to the heterogeneity seen in CBG. The latter is supported by the fact that desialated CBG migrates as two bands in IEF. Migration in IEF is based solely on charge, and since only sialic acid residues are charged in N-linked glycosylation, any heterogeneity seen for the desialated glycoprotein must reside within the protein moiety itself. The presence of O-glycosylation containing an N-acetylgalactosamine with a beta 1-3 linkage to galactose could not be demonstrated using O-glycosidase. N-terminal blockage could not account for the variation, as both the upper and lower variants were able to be sequenced resulting in identical sequences for the first 13 amino acids. The data presented supports the hypothesis that the differences in the sugar as well as the protein moiety are responsible for the heterogeneity seen for CBG.

Amidohydrolases↗
PubMed1999

Improved proton spectroscopic U-FLARE imaging for the detection of coupled resonances in the rat brain in vivo.

Modifications of the pulse sequence for spectroscopic U-FLARE imaging are discussed to detect not only the predominant singlet signals of N-acetylaspartate, total creatine, and choline containing compounds or the doublet signal of lactate, but also the coupled resonances of glutamate, glutamine, taurine and myo-inositol. Effective homonuclear decoupling is achieved by use of constant time chemical shift encoding. A maximum signal-to-noise ratio (SNR) can be obtained for a certain coupled resonance of interest by optimizing the evolution period t(c) of the J modulated spin echo. Good reproducibility and a high SNR were achieved by combining several methods for water suppression and by using the displaced variant of U-FLARE. Measurements of a 3 mm slice of the rat brain were performed in vivo within 4 min, giving a nominal voxel size of 1.5 x 1.5 x 3.0 mm3 or 1.5 x 0.75 x 3.0 mm3. Thus, optimized spectroscopic U-FLARE is a powerful tool for proton spectroscopic imaging with high spectral, spatial and temporal resolution.

Animals↗