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PubMed2005

Developing promiscuous glycosidases for glycoside synthesis: residues W433 and E432 in Sulfolobus solfataricus beta-glycosidase are important glucoside- and galactoside-specificity determinants.

Two residues that have been implicated in determining the substrate specificity of the thermophilic beta-glycosidase from the archaeon Sulfolobus solfataricus (SsbetaG), a member of the glycosyl hydrolase family 1, have been mutated by site-directed mutagenesis so as to create more versatile catalysts for carbohydrate chemistry. The wild-type and mutated sequences were expressed in E. coli with a His(7)-tag to allow one-step chromatographic purification. The E432C and W433C mutations removed key interactions with the OH-4 and OH-3 of the sugar substrates, thus reducing the discrimination of glucose, galactose and fucose with respect to other glycosides. This resulted in two glycosidases with greatly broadened substrate specificities. Observed changes include a 24-fold increase in Man:Gal activity and an 18-fold increase in GalA:Gal activity. This promiscuous substrate tolerance was further illustrated by the parallel synthesis of a beta-glycoside library of glucose, galactose, xylose and mannose in one pot at 50 degrees C, in organic solvent. The synthetic potential of the catalysts was further evaluated through alkyl glycoside transglycosylation yields, including the first examples of synthesis of beta-mannosides and beta-xylosides with SsbetaG.

Alkylation↗
PubMed2001

Flow cytometric DNA-quantification of three-color immunophenotyped cells for subpopulation specific determination of aneuploidy and proliferation.

A method is described for three-color immunophenotyping and simultaneous DNA-quantification using a flow cytometer equipped with a 488-nm argon laser and a mercury lamp (UV). The approach includes reproducible immunophenotyping comparing antigen expression before and after cell manipulation for DNA-measurement. The coefficients of variation after DNA-staining (CV=3.13 for T-cells in peripheral blood and CV=3.38 for T-cells in bone marrow) were adequate for exact DNA-analysis. For aneuploidy detection, a true internal standard was established measuring, for example, the DNA-content of T-cells in B-cell disease simultaneously with the DNA-content of the malignant cells. Using this method, aneuploidies could be unequivocally detected in 17 out of 24 patients with multiple myeloma. Furthermore, intratumor heterogeneities in DNA-content and antigen expression could be recognized, allowing an exact separation of tumor cells and normal hematopoiesis. The study also demonstrated the importance of exact immunophenotypic characterization of lymphocyte subpopulations and the determination of their specific proliferation, for example after proliferation induction in cell cultures. Future studies should address the applicability of this rather simple multiparameter approach for simultaneous immunophenotyping and DNA-measurement especially in the detection of minimal amounts of aneuploid cells after chemotherapy.

Aneuploidy↗
PubMed1996

Evaluations of enzyme-linked immunosorbent assay procedure for determining specific Epstein-Barr virus serology and of rapid test kits for diagnosis for infectious mononucleosis.

Using the results of Epstein-Barr virus-specific immunofluorescence serology as the "gold standard," we found that the sensitivities of the five rapid test kits varied from 78 to 84% and specificities varied from 89 to 100%. Enzyme-linked immunosorbent assay-determined specific Epstein-Barr virus antibody profiles had a sensitivity and specificity of 98.6 and 95.5%, respectively.

Antibodies, Viral↗
PubMedDate not supplied

Mitochondrial antibodies in primary biliary cirrhosis: species and nonspecies specific determinants of M2 antigen.

Sera from patients with primary biliary cirrhosis reacted with four major bands in beef heart mitochondria and ATPase extract when analyzed by immunoblot after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These four immunologically reactive bands corresponded to protein bands with molecular weights of about (a) 80,000; (b) 63,000; (c) 56,000; and (d) 43,000 to 46,000. An additional immunoreactive band was found with some high-titered primary biliary cirrhosis sera at 36,000. No association with any ATPase subunits was found, except for band c which migrated between the alpha- and beta-subunit of ATPase. Most ATPase fractions did not contain this band c, indicating that M2 determinants, as defined by immunoblot, are not identical with any ATPase subunit. Species and nonspecies-specific determinants of M2 were identified using mitochondria from rat liver and human heart and liver. Antigenic bands a, c and d were nonspecies-specific. Band b and e occurred only in beef heart. An additional determinant at about 38,000 was detected using human heart and liver mitochondria. Primary biliary cirrhosis sera showed a typical reaction with two protein bands of Escherichia coli, one at about 85,000 to 90,000 and the other at 60,000. Antibodies against both determinants could be absorbed with submitochondrial particles of beef heart showing that E. coli shares cross-reacting determinants with mitochondria. Sera from 56 primary biliary cirrhosis patients were tested using beef heart mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗
PubMedDate not supplied

[Specific determination of flaviviruses by molecular hybridization with synthetic deoxyoligonucleotide probes].

Molecular probes were designed for the purpose of specific determination of flavioviruses transmitted by the ticks of tick-borne encephalitis (TBE) and Omsk hemorrhagic fever (OHV) as well as by mosquitoes of Japanese encephalitis (JE), North Nile (NN), Murrey Valley encephalitis (MVE), Saint-Lois encephalitis (SLE), dengue 1-4 and of yellow fever (YF). The probes are synthetic deoxyoligonucleotides with the 18-20 long basis and complementary for the RNA fragments defined by computer analysis. The thus obtained probes, which specifically hybridize themselves with the sets of the TBE virus or of the OHV virus and do not hybridize themselves with other TBE viruses' sets. Group-specific probes for YE and dengue viruses as well as virus-specific probes, which are able to detect each of the above viruses without any cross effects, were suggested for indexing and identifying the flaviviruses transmitted by mosquitoes.

Encephalitis Viruses, Tick-Borne↗
PubMed2002

Using orthologous and paralogous proteins to identify specificity-determining residues in bacterial transcription factors.

Concepts of orthology and paralogy are become increasingly important as whole-genome comparison allows their identification in complete genomes. Functional specificity of proteins is assumed to be conserved among orthologs and is different among paralogs. We used this assumption to identify residues which determine specificity of protein-DNA and protein-ligand recognition. Finding such residues is crucial for understanding mechanisms of molecular recognition and for rational protein and drug design. Assuming conservation of specificity among orthologs and different specificity of paralogs, we identify residues that correlate with this grouping by specificity. The method is taking advantage of complete genomes to find multiple orthologs and paralogs. The central part of this method is a procedure to compute statistical significance of the predictions. The procedure is based on a simple statistical model of protein evolution. When applied to a large family of bacterial transcription factors, our method identified 12 residues that are presumed to determine the protein-DNA and protein-ligand recognition specificity. Structural analysis of the proteins and available experimental results strongly support our predictions. Our results suggest new experiments aimed at rational re-design of specificity in bacterial transcription factors by a minimal number of mutations.

Bacteria↗
PubMed1999

Specific determination of plasmin inhibitor activity in plasma: documentation of specificity of manual and automated procedures.

Recently, a chromogenic kit for determination of plasmin inhibitor activity, Coamatic Plasmin Inhibitor, has been developed with the aim to increase specificity and to allow application to a wide range of automates. The highly sensitive plasmin substrate S-2403 is used and, to further improve specificity, a low level of plasmin and a short incubation time is utilized as well as the incorporation of methylamine to quench alpha2-macroglobulin activity. The Coamatic Plasmin Inhibitor kit has been evaluated regarding specificity and compared with six commercially available plasmin inhibitor (also denoted alpha2-antiplasmin) kits and with the manual Immediate Plasmin Inhibition Test (IPIT) method, the reference method within the framework of the European Concerted Action against Thrombosis and Disabilities. Analysis of plasmin inhibitor deficient plasma with Coamatic Plasmin Inhibitor resulted in activities below 5% for all instrument applications, whereas all other commercial kits displayed unexpectedly higher values for this plasma (16-35%). There was a good agreement between the Coamatic Plasmin Inhibitor method and the IPIT method for all tested instrument applications; slope =0.80-1.02 and r = 0.92-0.97. No interference was detected from alpha2-macroglobulin, lysine, unfractionated and low-molecular weight heparin, and epsilon-amino caproic acid or from the non-plasminogen binding form of plasmin inhibitor.

Adult↗
PubMed1999

A sensitive and specific determination method for azaspiracids by liquid chromatography mass spectrometry.

A liquid chromatography/mass spectrometry (LC/MS) method was developed for the sensitive and specific determination of azaspiracid and its two analogs, the causative toxins of azaspiracid poisoning that occurred in the Netherlands and Ireland. The LC/MS method provided a detection limit of 50 pg for azaspiracid. The sensitivity was approximately 8 x 10(4) times greater than the mouse bioassay. The method was used to confirm the presence of azaspiracids in toxic mussels collected at Arranmore Island, Ireland in 1997.

Animals↗
PubMed1987

Specific determination and identification of cross-linked fibrin degradation products in patients under thrombolytic therapy for myocardial infarction.

We have demonstrated that the specific determination and identification of plasma FbDP alone is not sufficient to follow the effectiveness of thrombolytic therapy. Some patients who received an intravenous infusion of rt-PA for myocardial infarction had very high plasma FbDP levels, although no recanalization was observed angiographically. Since in many cases the FbDP levels increased after recanalization, it is assumed that rethrombosis may occur during and after t-PA treatment.

Antibodies, Monoclonal↗
PubMed2000

Specific determination of endothelial cell viability in the whole cell fraction from cryopreserved canine femoral veins using flow cytometry.

Abstract: An efficient method for specifically determining the viability of endothelial cells (EC) from cells dissociated from the human saphenous vein was investigated. Three different methods, trypan blue staining assay, [3H]-proline incorporation assay, and flow cytometry (FCM), combined with the fluorescein isothiocyanate conjugated with Griffonia simplicifolia agglutins (GS1-FITC)/propidium iodide (PI) double staining, were used. Both trypan blue staining and [3H] proline incorporation assays demonstrated less sensitivity to determine viability of EC differentially from the other cells. FITC-GS1 showed prominent binding to the vascular EC and could be counted by FCM including PI on dead cells. Following the cryopreservation process, the GS1-FITC/PI FCM analytical method was adopted to test simultaneously the viability of whole cells and EC from the same tissue, human saphenous veins, and mongrel dogs' femoral veins after harvesting, antibiotic solution treatment, and thawing. The viability of the whole cells from veins decreased with a significant difference (p < 0.05) from that of EC after thawing.

Animals↗
PubMed1978

Simultaneous and specific determination of proxyphylline, theophylline and other xanthine derivatives in serum by high pressure liquid chromatography.

A HPLC-method for rapid, simultaneous and specific determination of proxyphylline, theophylline, glyphylline, theobromine, caffeine and 3-methylxanthine using 8-chlorotheophylline as internal standard is presented. Using 20 microliter deproteinised serum samples for injection, the the detection limits for theophylline and proxyphylline are about 0.040 and 0.055 microgram per ml serum or plasma, respectively, and the corresponding sensitivities are about 0.033 and 0.048 microgram compound for 0.0044 absorbance units at 280 nm. In duplicate analyses the coefficient of variation is less than 3%. A number of frequently used drugs did not interfere in the analyses of serum samples from patients under treatment or in experiments with drug additions to serum samples.

Caffeine↗
PubMed1999

Identification of replication specificity determinants in two strains of tomato leaf curl virus from New Delhi.

We used two strains of tomato leaf curl virus from New Delhi to investigate specificity in replication of their cognate genomes. The strains share 94% sequence identity and are referred to as severe and mild on the basis of symptoms on tomato and tobacco. Replication assays in tobacco protoplasts and plants showed that a single amino acid change, Asn10 to Asp in the N terminus of Rep protein, determines specificity for replication of the two strains based upon its interaction with the origin of replication (ori) sequences. The change of Asp10 to Asn in Rep protein of the mild strain coupled with point mutations at the 3rd and 10th nucleotides of the 13-mer binding site altered its replication ability, resulting in increased levels of virus accumulation. Similarly, changing Asn10 to Asp in Rep protein of the severe strain impaired replication of the virus and altered its severe phenotype in plants. Site-directed mutations made in ori and Asn10 of Rep protein suggested that Asn10 recognizes the third base pair of the putative binding site sequence GGTGTCGGAGTC in the severe strain.

Asparagine↗
PubMed1975

Specific determination of testosterone in female plasma by radioimmunoassay: a rapid and reliable procedure for the routine clinical laboratory.

A rapid and reliable procedure for the specific determination of testosterone in female plasma is described. The assay entails a simple biphasic solvent partitioning of plasma extracts in order to remove cross-reacting steroids before quantitation of testosterone by radioimmunoassay using an antiserum raised against testosterone-11alpha-hemisuccinyl-bovine serum albumin. "Testosterone' values obtained in this way are in excellent agreeement with those obtained following thin-layer chromatographic purification of plasma extracts. However, the present procedure is easier, faster and cheaper to perform, and enables a single technician to assay 32 females plasma samples (in duplicate) in one working day.

Adrenal Glands↗
PubMed1994

A farnesylated domain in the G protein gamma subunit is a specific determinant of receptor coupling.

The interaction between receptor and a heterotrimeric G protein (alpha beta gamma) is thought to involve the intracellular loops of receptors and specific domains on the G protein. Here we show that a chemically farnesylated peptide (P5far) specific to the carboxyl-terminal domain (amino acids 60-71: DKNPFKELKGGC) of the gamma subunit of the G protein, Gt, directly stabilizes the active form of rhodopsin, metarhodopsin II (M II), and also uncouples rhodopsin-Gt interaction. Peptide activity is significantly affected by the absence of the isoprenoid moiety. Moreover, we show that altering the amino acid sequence of the farnesylated peptide by randomizing the sequence, substituting hydrophobic with hydrophilic residues (F64T; L67S) or deleting amino acids 60-66 significantly reduces the ability of the peptide to stabilize M II. This indicates that both the farnesyl moiety and the structure of the gamma subunit tail are specific determinants of receptor-G protein interaction. These results also suggest a general function for the family of G protein gamma subunits in signaling.

Amino Acid Sequence↗
PubMed2006

Keeping signals straight in transcription regulation: specificity determinants for the interaction of a family of conserved bacterial RNA-protein couples.

Regulatory systems often evolve by duplication of ancestral systems and subsequent specialization of the components of the novel signal transduction systems. In the Gram-positive soil bacterium Bacillus subtilis, four homologous antitermination systems control the expression of genes involved in the metabolism of glucose, sucrose and beta-glucosides. Each of these systems is made up of a sensory sugar permease that does also act as phosphotransferase, an antitermination protein, and a RNA switch that is composed of two mutually exclusive structures, a RNA antiterminator (RAT) and a transcriptional terminator. We have studied the contributions of sugar specificity of the permeases, carbon catabolite repression, and protein-RAT recognition for the straightness of the signalling chains. We found that the beta-glucoside permease BglP does also have a minor activity in glucose transport. However, this activity is irrelevant under physiological conditions since carbon catabolite repression in the presence of glucose prevents the synthesis of the beta-glucoside permease. Reporter gene studies, in vitro RNA-protein interaction analyzes and northern blot transcript analyzes revealed that the interactions between the antiterminator proteins and their RNA targets are the major factor contributing to regulatory specificity. Both structural features in the RATs and individual bases are important specificity determinants. Our study revealed that the specificity of protein-RNA interactions, substrate specificity of the permeases as well as the general mechanism of carbon catabolite repression together allow to keep the signalling chains straight and to avoid excessive cross-talk between the systems.

Bacillus subtilis↗
PubMed1996

Golgi apparatus mammary gland casein kinase: monitoring by a specific peptide substrate and definition of specificity determinants.

The casein kinase from the Golgi apparatus of lactating mammary gland (GEF-CK) is distinct from ubiquitous 'casein kinases' termed protein kinases CK1 and CK2 and appears to define a family of secretory pathways protein kinases that phosphorylate seryl residues followed by an acidic residue at position +2. In this report we show that a new synthetic peptide substrate derived from beta-casein (beta[28-40]) is suitable for the fast, efficient and selective monitoring of GEF-CK, being unaffected by CK1 and CK2, and we define the consensus sequence of this protein kinase as being Ser-Xaa-Glu/SerP, distinct from that of CK2 (Ser/Thr-X-X-Glu/Asp/SerP/TyrP). In particular, the failure to recognize Asp as crucial specificity determinant prevents the phosphorylation of the specific CK2 peptide substrate RRRADDSDDDDD by GEF-CK. Thus, peptide substrates are now available for the fast and specific monitoring of all the three classes of 'casein kinases', CK1, CK2 and GEF-CK.

Amino Acid Sequence↗
PubMed1992

[Immunoenzyme analysis of antigen-specific determination of HBsAG-containing circulating immune complexes (CIC HBsAG/IgM and CIC HBsAG/IgG) in blood serum of patients with HBV-infection].

A complex enzyme immunoassay (ELISA) has been designed for antigen-specific determination of HBsAg-containing circulating immune complexes (CIC HBsAg/IgM and CIC HBsAg/IgG) in human blood sera in parallel with registration of free HBsAg and specific antibodies to viruses of hepatitis A, B and D. It is shown that effective formation of HBsAg-containing CIC serologically is registered predominantly as a mutually incompatible marker with detection of free HBsAg (in 70-85% of the cases). CIC HBsAg/IgM and CIC HBsAg/IgG may be registered both in parallel and as mutually exclusive markers. Effective formation of HBsAg-containing CIC in the presence of anti-HBsAg occurs in case of a mild course of viral hepatitis of epidemic and sporadic type, while in severe forms of VH-free HBsAg is predominantly detected thus pointing either to ineffective formation of HBsAg-containing CIC or to their continuous registration with demonstration of the effect of delay of witching of anti-HBsM over to anti-HBsG (or CIC HBsAg/IgM to CIC HBsAg/IgG). It was also found that in case of epidemic VH in Tajik SSR (1987) serologically marked as VH both A and B convalescent phase was characterized by parallel disappearance (or lowering of the titer levels) of HBsAg-containing CIC and class M antibodies to both hepatitis A (anti-HAV M) and B (anti-HBcM, anti-HBsM) along with the containing parallel registration of relevant G-antibodies (anti-HAV G/anti-HBcG). This observation requires further studies both in terms of close association of viruses of hepatitides A and B and with regards to possible antigenic mimicry.

Adult↗
PubMed2000

Plasticity in the primary binding site of galactose/N-acetylgalactosamine-specific lectins. Implication of the C-H...O hydrogen bond at the specificity-determining C-4 locus of the saccharide in 4-methoxygalactose recognition by jacalin and winged bean (basic) agglutinin I.

It is currently believed that an unsubstituted axial hydroxyl at the specificity-determining C-4 locus of galactose is indispensable for recognition by galactose/N-acetylgalactosamine-specific lectins. Titration calorimetry demonstrates that 4-methoxygalactose retains binding allegiance to the Moraceae lectin jacalin and the Leguminosae lectin, winged bean (basic) agglutinin (WBA I). The binding reactions were driven by dominant favorable enthalpic contributions and exhibited significant enthalpy-entropy compensation. Proton NMR titration of 4-methoxygalactose with jacalin and WBA I resulted in broadening of the sugar resonances without any change in chemical shift. The alpha- and beta-anomers of 4-methoxygalactose were found to be in slow exchange with free and lectin-bound states. Both the anomers experience magnetically equivalent environments at the respective binding sites. The binding constants derived from the dependence of NMR line widths on 4-methoxygalactose concentration agreed well with those obtained from titration calorimetry. The results unequivocally demonstrate that the loci corresponding to the axially oriented C-4 hydroxyl group of galactose within the primary binding site of these lectins exhibit plasticity. These analyses suggest, for the first time, the existence of C-H.O-type hydrogen-bond(s) in protein-carbohydrate interactions in general and between the C-4 locus of galactose derivative and the lectins jacalin and WBA I in particular.

Acetylgalactosamine↗