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At least 271 records · Page 15Linked to original sources
PubMed2001

An integrated restriction fragment length polymorphism--amplified fragment length polymorphism linkage map for cultivated sunflower.

Restriction fragment length polymorphism (RFLP) maps have been constructed for cultivated sunflower (Helianthus annuus L.) using three independent sets of RFLP probes. The aim of this research was to integrate RFLP markers from two sets with RFLP markers for resistance gene candidate (RGC) and amplified fragment length polymorphism (AFLP) markers. Genomic DNA samples of HA370 and HA372, the parents of the F2 population used to build the map, were screened for AFLPs using 42 primer combinations and RFLPs using 136 cDNA probes (RFLP analyses were performed on DNA digested with EcoRI, HindIII, EcoRV, or DraI). The AFLP primers produced 446 polymorphic and 1101 monomorphic bands between HA370 and HA372. The integrated map was built by genotyping 296 AFLP and 104 RFLP markers on 180 HA370 x HA372 F2 progeny (the AFLP marker assays were performed using 18 primer combinations). The HA370 x HA372 map comprised 17 linkage groups, presumably corresponding to the 17 haploid chromosomes of sunflower, had a mean density of 3.3 cM, and was 1326 cM long. Six RGC RFLP loci were polymorphic and mapped to three linkage groups (LG8, LG13, and LG15). AFLP markers were densely clustered on several linkage groups, and presumably reside in centromeric regions where recombination is reduced and the ratio of genetic to physical distance is low. Strategies for targeting markers to euchromatic DNA need to be tested in sunflower. The HA370 x HA372 map integrated 14 of 17 linkage groups from two independent RFLP maps. Three linkage groups were devoid of RFLP markers from one of the two maps.

Chromosome Mapping↗
PubMedDate not supplied

[Molecular typing by amplified fragment length polymorphism and PCR-restriction fragment length polymorphism , biotyping and antimicrobial susceptibility of Campylobacter jejuni].

Molecular typing systems have provided invaluable information for tracking infectious agents through the food chain. These tools have been essential for understanding the epidemiology of gastrointestinal infectious diseases, therefore providing essential and evidence-based information for appropriate interventions and preventative measures. Two such molecular typing techniques based on the polymerase chain reaction (PCR) that have been applied to the epidemiology of foodborne pathogens are, amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) analysis. Campylobacter is responsible for one of the most common bacteria foodborne gastrointestinal infections affecting humans, especially in developed countries. The object of this paper is to apply AFLP and RFLP analysis of the flagellin (flaA) gene to 18 isolates of Campylobacter jejuni from human sporadic cases in Italy. Results of these analyses were compared to the phenotypes of these isolates based on biotyping and antimicrobial resistance determinations. All isolates were typable by the four methods. The RFLP procedure was performed with DdeI and HinfI enzymes, and 12 and 8 distinct profiles respectively were recognised. AFLP analysis was more discriminatory, and recognised 16 different profiles. Results from AFLP were reproducible and applicable for definitive characterisation of C. jejuni isolated from different outbreaks. PCR-RFLP of the flaA gene represents a useful tool only to compare isolates within a single outbreak.

Anti-Bacterial Agents↗
PubMed1976

Formation of three-dimensional structure in protein fragments. Reactivation of reduced hen egg lysozyme fragment 1-127.

Regeneration of enzymic activity from reduced hen egg lysozyme peptide 1-127 was effected with a glutathione oxidation-reduction buffer. The rate of regeneration was nearly as great for peptide 1-127 as for reduced lysozyme itself, and the yields were the same (greater than 80%). The regenerated fragment 1-127 was shown to be indistinguishable from fragment 1-127 before reduction by ion exchange chromatography, amino acid analysis, polyacrylamide gel electrophoresis, and disulfide analysis. These results show that the COOH-terminal dipeptide Arg-Leu is not essential for the acquisition of the native three-dimensional structure of lysozyme.

Amino Acids↗
PubMed1992

[Conformational aspects of the biological effect of functional fragments of the p21ras oncoprotein family. 2. Fragment 1-16, various sequences].

The conformational analysis data on active ([Val12-Gly13], [Asp12-Gly13] and [Gly12-Asp13]) and passive ([Gly12-Gly13] and [Pro12-Gly13]) modifications of the p21ras family oncoproteins are presented. The activating amino acid substitutions are shown to be accompanied by essential changes in the secondary structure, resulted in the 9-16 fragment spiralization. The spatial structure of the 1-9 fragment does not vary for all the predominant forms of the active and passive analogues. The results of the conformational analysis have been used for studying the structural-functional relationships.

Amino Acids↗
PubMed1977

Artificial hybrid protein containing a toxic protein fragment and a cell membrane receptor-binding moiety in a disulfide conjugate. I. Synthesis of diphtheria toxin fragment A-S-S-human placental lactogen with methyl-5-bromovalerimidate.

In order to study the mechanism of entry of plant seed and bacterial toxins into mammalian cells, methods have been developed to synthesize artificial protein hybrid conjugates containing a moiety which binds to a cell membrane receptor and an active fragment of a toxin protein. Utilizing methyl-5-bromovalerimidate, a disulfide cross-linked conjugate of human placental lactogen (hPL) and diphtheria toxin fragment A (toxin A) was synthesized. The reagent was prepared from 5-bromovaleryl nitrile by Pinner synthesis and then used to amidinate hPL. The bromo group thus introduced was converted to S-sulfonate by nucleophilic displacement with 1 M aqueous sodium thiosulfate at room temperature overnight. The S-sulfonated hPL reacted readily with the-SH gorup of reduced toxin A to form a 1 mol/mol of disulfide conjugate in high yield. Thus when reduced toxin A was incubated with a 4-fold excess of the hPL S-sulfonate at 4 degrees and pH 6.5 for 120 h, a conjugate yield of 50% relative to the toxin A input was obtained. Homopolymer formation was negligible and the product was purified by gel filtration on Sephadex G-150. Purity of the conjugate estimated by quantitative analysis of sodium dodecyl sulfate gels was 90%. The toxin A-hPL conjugate retained the activities of both toxin A and hPL, as reported in the accompanying paper. This method of preparing protein hybrid conjugates appeared to have advantages over previous methods utilizing bifunctional reagents with respect to both yield and freedom from homopolymer formation.

Animals↗
PubMed1979

The amino acid sequence of fragment A, an enzymically active fragment of diphtheria toxin. II. The cyanogen bromide peptides.

Cyanogen bromide cleavage of Fragment A from diphtheria toxin at the four methionines present in each molecule resulted in five major peptides which were isolated and studied by sequence methods. These five peptides of 4, 11, 14, 63, and 101 residues account for all 193 residues in Fragment A and provide overlaps for the tryptic peptides from the maleylated protein. Two additional peptides were isolated and shown to be shorter forms (8 and 10 residues) of the COOH-terminal cyanogen bromide peptide (11 residues).

Adenosine Diphosphate Sugars↗
PubMed1980

Polyclonal activation of murine B lymphocytes by Fc fragments. III. Characterization of the defect in the ability of the C3H/HeJ mouse to respond to Fc fragments.

Spleen cell preparations from the C3H/HeJ mouse were found to be refractory to Fc fragment-induced polyclonal activation. This defect was not a characteristic of all C3H strains, because spleen cells from the histocompatible C3Heb/FeJ strain produced polyclonal antibody in the presence of Fc fragments. The cellular defect was found to be associated with the macrophage population. Macrophages from C3H/HeJ mice were incapable of generating mitogenic Fc subfragments from intact Fc, whereas C3Heb/FeJ performed this function normally. That T cells and B cells from the C3H/HeJ mouse were normal was shown by the fact that C3H/HeJ spleen cells responded to Fc subfragments. Moreover, T cells from C3H/HeJ spleen cell populations could be substituted for C3Heb/FeJ-derived T cells.

Animals↗
PubMed1994

Nondetectability of restriction fragments and independence of DNA fragment sizes within and between loci in RFLP typing of DNA.

We provide experimental evidence showing that, during the restriction-enzyme digestion of DNA samples, some of the HaeIII-digested DNA fragments are small enough to prevent their reliable sizing on a Southern gel. As a result of such nondetectability of DNA fragments, individuals who show a single-band DNA profile at a VNTR locus may not necessarily be true homozygotes. In a population database, when the presence of such nondetectable alleles is ignored, we show that a pseudodependence of alleles within as well as across loci may occur. Using a known statistical method, under the hypothesis of independence of alleles within loci, we derive an efficient estimate of null allele frequency, which may be subsequently used for testing allelic independence within and across loci. The estimates of null allele frequencies, thus derived, are shown to agree with direct experimental data on the frequencies of HaeIII-null alleles. Incorporation of null alleles into the analysis of the forensic VNTR database suggests that the assumptions of allelic independence within and between loci are appropriate. In contrast, a failure to incorporate the occurrence of null alleles would provide a wrong inference regarding the independence of alleles within and between loci.

Alleles↗
PubMed1993

[Evaluation of an enzyme-linked immunosorbent assay for the determination of prothrombin fragment F1.2 (Dade Prothrombin Fragment F1.2 ELISA: Baxter Diagnostics Inc., U.S.A.) using micro-titer plate].

Prothrombin fragment F1.2 (F1.2) is a new molecular marker indicating acceleration of blood coagulation. We evaluated a new assay of F1.2 measurement using a micro-titer plate (Dade Prothrombin Fragment F1.2 ELISA: Baxter Diagnostics Inc., U.S.A.). The assay obtained satisfactory results in intra-assay reproducibility test, inter-assay reproducibility test, dilution linearity test and in vitro recovery test. Normal values of plasma F1.2 were 0.16 +/- 0.09 nmol/l (mean +/- SD) in 108 healthy individuals. Differences in the levels between the sexes were not significant. In patients with DIC (n = 22), plasma F1.2 levels were significantly higher than in normal healthy individuals and were correlated with the levels of thrombin-antithrombin III complex. These findings suggest that this F1.2 assay using a micro-titer plate is clinically useful for the evaluation of the therapeutic effect and diagnosis of hypercoagulable states like DIC.

Adolescent↗
PubMed1993

Recombinant human antibodies: linkage of an Fab fragment from a combinatorial library to an Fc fragment for expression in mammalian cell culture.

The combinatorial phage library approach to immunoglobulin repertoire cloning recently made it possible to isolate gene fragments encoding human immunoglobulin G1 Fabs binding with high affinity to specific antigens. Here we describe the construction of genes encoding whole human anti-tetanus toxoid antibodies based on one of these gene fragments and the efficient expression of these constructs by co-transfection of separate heavy and light chain vectors into a Chinese hamster ovary cell line constitutively expressing a viral transactivator protein. This system will be generally useful for the rapid analysis of recombinant antibodies derived from combinatorial libraries.

Amino Acid Sequence↗
PubMed1989

Transformation of NIH 3T3 cells by herpes simplex type 2 BglII n fragment and sub-fragments is independent from induction of mutation at the HPRT locus.

The effect of transfection of the herpes simplex virus type 2 transforming fragment BglII n and of its three Xhol subfragments on mutagenesis and morphological transformation was assayed in NIH 3T3 cells. While BglII n and the right hand portion of this fragment increased the number of transformed foci, no significant effects on the mutation frequency at the hprt locus were observed. Our results indicate that transformation by BglII n is independent from the induction of somatic mutations and suggest that other mechanisms must be considered to explain transformation by this sequence.

Animals↗
PubMed2005

Purification and characterization of a recombinant anti-angiogenic kringle fragment expressed in Escherichia coli: Purification and characterization of a tri-kringle fragment from human apolipoprotein (a) (kringle IV (9)-kringle IV (10)-kringle V).

A kringle fragment (type IV (9)-IV (10)-V) from human apolipoprotein (a) (called LK68) was expressed in an inclusion body in Escherichia coli. The LK68 in this inclusion body was rendered soluble with urea, and efficiently refolded via oxidation in the presence of re-dox couple. The refolded LK68 was then purified via two steps of ion exchange chromatography, concentrated via preparative reversed-phase chromatography, and freeze-dried, at a final yield of approximately 30%. The purified LK68 exhibited profound affinity for lysine and fibrinogen, which suggests the proper folding of the kringle fragment, and also indicates that the native characteristics of apolipoprotein (a) were preserved. The purified LK68 was determined to be highly homogeneous upon reversed-phase HPLC analysis and size-exclusion HPLC analysis, in the presence of 20% (v/v) acetonitrile. However, on size-exclusion HPLC analysis without acetonitrile, it was determined to be somewhat heterogeneous, and this was corroborated by native analyses, including native PAGE and IEF.

Angiogenesis Inhibitors↗
PubMed2002

High performance liquid chromatography of phenolic choline ester fragments derived by chemical and enzymatic fragmentation processes: analysis of sinapine in rape seed.

High-performance liquid chromatography methods based on reversed-phase chromatography (RPC) and normal phase chromatography (NPC) were introduced for the separation of some representative phenolic acids, choline and betaine, which are the fragments of phenolic choline esters. Sinapine, which is the major phenolic choline ester found in rape seed, was quantitatively hydrolyzed to choline and sinapic acid upon treatment with a solution of sodium hydroxide at room temperature. Choline was further converted to betaine by incubating the base hydrolyzate with choline oxidase. Both sinapic acid and betaine formed the basis for the quantitative determination of sinapine in rape seed by RPC and NPC, respectively. The amounts of sinapine found in rape seed via either of the two fragments (i.e., sinapic acid or betaine) were in very close agreement.

Betaine↗
PubMedDate not supplied

[Synthesis, NMR and conformational studies of fucoidan fragments. VI. Fragments, content of alpha-(1--->2)-bound fucobioside unit].

A series of selectively sulfated di- and trisaccharide derivatives corresponding to the potential fragments of fucoidans with a (1-->2)-alpha-bound fucobioside unit were synthesized and studied by 1H and 13C NMR spectroscopy. NOE experiments and molecular modeling were used for a conformational analysis of the compounds synthesized. In the case of disaccharides, the experimental NOE values were found to agree with those obtained using modeling with the use of density functional theory (DFT) and differ from those resulting from modeling by the molecular mechanics MM3 force field. Trisaccharide fragments partially or completely sulfated in position 4 turned out to be correctly described by both MM3 force field and DFT computation. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru.

Magnetic Resonance Spectroscopy↗
PubMed1981

Sequencing long DNA fragments cloned in bacteriophage M13 by using internal primers. The sequence analysis of a yeast DNA fragment containing a replication origin.

In the ;shotgun' procedure for sequencing DNA, DNA fragments are cloned into a phage M13 vector and sequenced by using a flanking primer. In a variation of this procedure a longer DNA sequence is cloned into M13, the two single-stranded recombinants identified and sequenced by using a set of internal primers prepared by exonuclease III digestion of restriction fragments.

Base Sequence↗
PubMed1995

Structure of heparin fragments with high affinity for lipoprotein lipase and inhibition of lipoprotein lipase binding to alpha 2-macroglobulin-receptor/low-density-lipoprotein-receptor-related protein by heparin fragments.

Heparin-derived deca- and octa-saccharides were subjected to affinity chromatography on lipoprotein lipase-Sepharose and the fractions eluted at high salt concentration were analysed by strong-anion-exchange chromatography. Two high-affinity decasaccharides were isolated and the structure determined by one- and two-dimensional 1H-n.m.r. spectroscopy. The affinities of 3H-labelled low-molecular-mass heparin and size-fractionated deca-, octa-, and hexa-saccharides for lipoprotein lipase immobilized on microtitre plates were determined from saturation curves. From competition experiments the affinities of unlabelled heparins and pure deca- and hexa-saccharide fragments were determined. The binding was size- and charge-dependent, but structural dependency was also indicated. Thus substitution of a 2-O-sulphated L-iduronic acid with D-glucuronic acid was less important than the sulphation pattern of the D-glucosamine residue for affinity for lipoprotein lipase. Heparin inhibits binding of lipoprotein lipase to alpha 2-macroglobulin-receptor/low-density-lipoprotein receptor-related protein. The effects of size, charge and structure for this inhibition were studied. The ability of the heparin fragments to inhibit binding correlated with their affinity for lipoprotein lipase. This indicates that the inhibition of the binding of lipoprotein lipase to alpha 2-macroglobulin-receptor/low-density-lipoprotein receptor-related protein by heparin is exclusively mediated by binding of heparin to lipoprotein lipase.

Amino Acid Sequence↗
PubMed2005

Fragmentation dynamics of fullerenes in intense femtosecond-laser fields: loss of small neutral fragments on a picosecond time scale.

The fragmentation dynamics of C60 irradiated with intense femtosecond laser pulses is studied with one-color pump-probe spectroscopy. Small neutral fragments (C, C2, and C3) are formed by an 800-nm pump pulse which are then postionized by a delayed probe pulse. The respective ion signals detected by the time-of-flight mass spectrometry dramatically increase on a time scale of 10-20 ps.

Journal Article↗
PubMed2000

The destabilization of lipid membranes induced by the C-terminal fragment of caspase 8-cleaved bid is inhibited by the N-terminal fragment.

Bid is a proapoptotic, BH3-domain-only member of the Bcl-2 family. In Fas-induced apoptosis, Bid is activated through cleavage by caspase 8 into a 15.5-kDa C-terminal fragment (t(c)Bid) and a 6.5 kDa N-terminal fragment (t(n)Bid). Following the cleavage, t(c)Bid translocates to the mitochondria and promotes the release of cytochrome c into the cytosol by a mechanism that is not understood. Here we report that recombinant t(c)Bid can act as a membrane destabilizing agent. t(c)Bid induces destabilization and breaking of planar lipid bilayers without appearance of ionic channels; its destabilizing activity is comparable with that of Bax and at least 30-fold higher than that of full-length Bid. Consistently, t(c)Bid, but not full-length Bid, permeabilizes liposomes at physiological pH. The destabilizing effect of t(c)Bid on liposomes and planar bilayers is independent of the BH3 domain. In contrast, mutations in the BH3 domain impair t(c)Bid ability to induce cytochrome c release from mitochondria. The permeabilizing effect of t(c)Bid on planar bilayers, liposomes, and mitochondria can be inhibited by t(n)Bid. In conclusion, our results suggest a dual role for Bid: BH3-independent membrane destabilization and BH3-dependent interaction with other proteins. Moreover, the dissociation of Bid after cleavage by caspase 8 represents an additional step at which apoptosis may be regulated.

Animals↗