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PubMed1989

Blood clotting factor IX BM Nagoya. Substitution of arginine 180 by tryptophan and its activation by alpha-chymotrypsin and rat mast cell chymase.

Factor IX BM Nagoya (IX Nagoya) is a natural mutant of factor IX responsible for severe hemophilia B. A patient with this mutant is characterized by a markedly prolonged ox brain prothrombin time. IX Nagoya was purified from the patient's plasma by immunoaffinity chromatography with an anti-factor IX monoclonal antibody column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that treatment of IX Nagoya with factor XIa/Ca2+ resulted in cleavage only at the Arg145-Ala146 bond. Reversed-phase high performance liquid chromatography of a trypsin digest of IX Nagoya showed an aberrant peptide, which was further digested with proteinase Asp-N. Primary structure analysis of one of the Asp-N peptides revealed that Arg180 is replaced by Trp. An essentially complete (99%) amino acid sequence of IX Nagoya was obtained by sequencing fragments derived from a lysyl endopeptidase digest in which no other substitutions in the catalytic triad or substrate binding site were found. We also found that IX Nagoya is activated by alpha-chymotrypsin or rat mast cell chymase by monitoring the rate of factor X activation using a fluorogenic peptide substrate in the presence of factor VIII, phospholipids, and Ca2+. These results indicate that the substitution of Arg180 by Trp impairs the cleavage by factor XIa required for activation of this zymogen and that the substitution causes hemophilia BM.

Amino Acid Sequence↗

PubMed1985

Conformational studies of two histamine H2-receptor antagonistic phenylformamidines: mifentidine and its guanidinothiazole analogue DA 4643.

Two histamine H2-receptor antagonists of the phenylformamidine type, mifentidine (N-isopropyl-N'-(4-1H-imidazol-4-yl-phenyl) formamidine dihydrochloride; I) and DA 4643 (N-methyl-N'-(3-(2-guanidinothiazol-4-yl)-phenyl) formamidine dihydrochloride; II), have been investigated by experimental physico-chemical studies and theoretical conformational analysis. PKa determinations on the two molecules I and II show that these substances exist at physiological pH (7.4) predominantly as their monoprotonated forms at the formamidine moiety. Semiempirical quantum mechanical (MNDO, CNDO/2) and molecular mechanics (MMPI) calculations show a preference of the nearly planar conformations for I and of different low energy rotamers for II. The energy of these conformers is a function of two important torsion angles, one around the bond joining the imidazole, or the guanidinothiazole, and the phenyl ring and the other around the bond joining the phenyl ring and the formamidinium cation. When the distances between crucial parts present in I and II are considered, it results that the relatively higher flexibility of II allows accommodation of amidine pairs present in the latter at a distance similar to that found for correspondent pairs in the conformationally more restricted I. Conformational aspects of I and II are discussed with reference to a recently described conformation of cimetidine determined by X-ray method. A hypothesis of binding of H2-receptor antagonists of the phenylformamidine type is advanced with reference to electrostatic potential maps calculated for crucial part structures of I, II and cimetidine. The present work supports the hypothesis that both mifentidine and DA 4643 interact with the histamine H2-receptor at the same site, utilizing in the binding process the same, or closely similar, receptor structural features.

Chemical Phenomena↗

PubMed1985

Multidirectional tomography in otospongiosis.

Eight clinical situations are best examined by polytomography, namely, ossicular chain disruption, malleus fixation, assessing the extent of footplate fixation, delineating cochlear otospongiosis, postoperative studies of unsuccessful cases, developing an anatomic map preoperatively with particular reference to the intratympanic course of the facial nerve, ruling in or out the rare closure of the round window by otospongiosis, and differentiation of the congenital footplate fixation from true otospongiosis. We discuss each of these situations briefly.

Cochlea↗

PubMed1983

The architecture of the animal fatty acid synthetase. I. Proteolytic dissection and peptide mapping.

The vertebrate fatty acid synthetase complex consists of two, apparently identical, subunits which contain seven catalytic centers and an acyl carrier site. As a prelude to studying the functional organization of this system, a comprehensive proteolytic analysis of the chicken fatty acid synthetase subunit has been carried out. Of 24 proteases tested, seven (chymotrypsin, elastase, trypsin, Myxobacter protease, subtilisins A and B, and kallikrein) were found to be capable of inflicting specific cleavages in the native enzyme. The proteolytic profiles were analyzed with respect to both the kinetics and the size of the fragments generated. The relationships between the various fragments were further delineated by combinatorial digestions. All of the data points to the fact that the subunits are indeed identical. The cleavage patterns indicate that the fatty acid synthetase subunit is primarily arranged into three major domains of Mr 127,000, 107,000, and 33,000, since these fragments are commonly produced by most of the proteases. The latter peptides may be further subdivided and mapped by the action of individual enzymes into a number of distinct regions: the 127,000 domain into fragments of Mr 60,000, 45,000, and 23,000; the 107,000 domain into fragments of Mr 14,000, 56,000, 21,000, and 15,000; all aligned accordingly. This proteolytic map serves as a reference for the controlled dissection of the fatty acid synthetase complex, thereby allowing determination of the location of functional centers within the parent polypeptide.

Animals↗

PubMed1971

Localization of a portion of the ribosomal RNA genes in Escherichia coli.

About one-half of the genes coding for ribosomal RNA in Escherichia coli have been localized by the use of F-merogenotes. These cistrons have been positioned between 74 and 77 min on the E. coli map described by Taylor and Trotter. The episomal DNA carrying these genes was enriched by chromatography. The map position is discussed with reference to previous localization attempts.

Centrifugation, Density Gradient↗

PubMed1988

Mapping of event-related desynchronization and type of derivation.

Event-related desynchronization (ERD) is a term describing alpha band amplitude changes in response to an event (stimulus presentation, self-paced movement, etc.). ERD mapping is a brain-imaging technique used to display the topographical pattern and time course of alpha power changes. Multi-lead EEG data referred to one ear were recorded during voluntary finger movements. From these data, transverse bipolar, source and common average reference derivations and the laplacian operator were calculated, and ERD maps are computed. The ERD is enhanced and best localized with the laplacian operator method, or with source derivations. ERD maps with ear reference required cautious interpretation.

Alpha Rhythm↗

PubMed1991

Use of high coverage reference libraries of Drosophila melanogaster for relational data analysis. A step towards mapping and sequencing of the genome.

Three differently made, primary Drosophila cosmid libraries of 16-fold genome coverage have been generated. Also, a jumping library has been created by a new method that takes advantage of methylation differences between genomic DNA and vector. Thirdly, two cDNA libraries have been picked. All these libraries have been arrayed on high-density in situ filters, each containing 9216 clones. As a reference system, such filters are distributed and identified clones are provided. Single-copy probes have identified on average 1.4 cosmids per genome equivalent. Together with cytogenetically mapped yeast artificial chromosomes, the libraries are also being used for physically mapping the genome, mainly by oligonucleotide fingerprinting and pool hybridizations. cDNA clones are further examined by a partial sequencing analysis by oligomer hybridization.

Animals↗

PubMed1992

Three-dimensional whole-brain mapping.

The implementation and use of a computerized whole-brain mapping system which can be used in conjunction with MRI, CT, angiographic and other brain imaging techniques is described. Three mapping systems based upon common reference structures about the third and fourth ventricular core of the brain are used in conjunction with internationally recognized nomenclature to create a normalized whole-brain mapping system according to the Talairach/Tournoux proportional grid technique.

Brain↗

PubMed1990

A genetic linkage map of chromosome 17.

We have developed a genetic linkage map of 19 markers (including nine genes) on human chromosome 17, providing 13 reference points along virtually the entire length of this chromosome. The map covers an estimated 149 cM in length (sex-averaged), with a total length of 214 cM in females and 95 cM in males. This sex difference appears to be significant along virtually the entire length of the map. This map will be useful both for providing reference points for fine structure genetic and physical mapping and for genetic linkage studies of diseases, including von Recklinghausen neurofibromatosis and Charcot-Marie-Tooth disease.

Chi-Square Distribution↗

PubMed1989

Perceptual and conceptual factors in distortions in memory for graphs and maps.

We propose that representations of visual stimuli are a consequence of both perceptual and conceptual factors that may be revealed in systematic errors in memory. Three experiments demonstrated increased (horizontal or vertical) symmetry in perception and memory of nearly symmetric curves in graphs and rivers in maps. Next, a conceptual factor, an accompanying description biasing toward symmetry or asymmetry, also distorted memory in the expected direction for the symmetric descriptions. In the two final experiments, we investigated conceptual factors in selection of a frame of reference. Subjects remembered lines in graphs, but not in maps, as closer to the imaginary 45 degrees line. Combined with earlier research, this suggests that the reference frame for map lines is the canonical axes and for graph lines, the imaginary 45 degrees line.

Adult↗

PubMed1977

Human papillomavirus DNA: physical mapping of the cleavage sites of Bacillus amyloliquefaciens (BamI) and Haemophilus parainfluenzae (HpaII) endonucleases and evidence for partial heterogeneity.

The DNA of human papillomavirus (HPV) obtained from a pool of plantar warts is cleaved by bacillus amyloliquefaciens (BamI) and Haemophilus parainfluenzae (HpaII) restriction endonucleases at one and four specific sites, respectively. These sites were localized on the previously established cleavage map of HPV DNA, using the Hind, HindIII, HpaI, and EcoRI endonuclease restriction sites as reference. The four HpaII sites were mapped, clockwise, at 1.4, 41.1, 44.3, and 52.8% of the genome length from the unique BamI cleavage site taken as point zero. The HpaII site mapped at 1.4% of the genome length was absent in 40 to 50% of the molecules, thus showing a genetic heterogeneity of HPV DNA.

Bacillus↗

PubMed1990

An intron mutation in the human alpha 1(I) collagen gene alters the efficiency of pre-mRNA splicing and is associated with osteogenesis imperfecta type II.

This study describes a homozygous, G----A transition at the moderately conserved +5 position within the splice donor site of intron 14 in the human alpha 1(I) collagen gene. The mutation reduced the efficiency of normal splice-site selection since the exon upstream of the mutation was spliced alternatively. Moreover, the extent of alternative splicing was sensitive to the temperature at which the mutant cells were grown, suggesting that the mutation directly affected spliceosome assembly. To achieve exon skipping, this effect must be propagated so as to disrupt the selection of a second splice site in the adjacent intron.

Adenine↗

PubMed1974

Specification of positional information in retinal ganglion cells of Xenopus laevis: intra-ocular control of the time of specification.

Programming events in the stage 28-31 clawed frog embryo partly determine the organization of the future retinotectal map by specifying the permanent reference axes for cellular positional information in the retina. Thus, when transplanted in 180 degrees -rotated orientation into a stage 27/28 orbit, an unspecified (stage 28 or younger) eye can acquire new axes and develop a normally-oriented retinotectal map, whereas a specified (stage 31 or older) eye retains its original axes and develops an inverted map. We have used the retinotectal map to determine when (and under what conditions) specification with changes in orientation occurred in eye primordia serially transplanted between embryos of different stages. Specification was not precipitated when stage 22/23 eyes were grafted into stage 28-32 embryos or explanted in vitro, nor was specification delayed when stage 28 eyes were grafted into embryos younger than stage 28. Control experiments confirmed the general correlation between the time of specification and the stage of the eye primordium. We infer that intraocular regulatory mechanisms control the time of specification.

Animals↗

PubMed2025

Reference genome bias in light of species-specific chromosomal reorganization and translocations.

BACKGROUND: Whole-genome sequencing efforts, have during the past decade, unveiled the central role of genomic rearrangements-such as chromosomal inversions-in evolutionary processes, including local adaptation in a wide range of taxa. However, employment of reference genomes from distantly or even closely related species for mapping and the subsequent variant calling can lead to errors and/or biases in the datasets generated for downstream analyses. RESULTS: Here, we capitalize on the recently generated chromosome-anchored genome assemblies for Arctic cod (Arctogadus glacialis), polar cod (Boreogadus saida), and Atlantic cod (Gadus morhua) to evaluate the extent and consequences of reference bias on population sequencing datasets (approx. 15-20 × coverage) for both Arctic cod and polar cod. Our findings demonstrate that the choice of reference genome impacts the mapping statistics, including mapping depth and mapping quality, as well as core population genetic estimates, such as heterozygosity levels, nucleotide diversity (π), and cross-species genetic divergence (DXY). Furthermore, using a more distantly related reference genome can lead to inaccurate detection and characterization of chromosomal inversions, i.e., in terms of size (length) and location (position), due to inter-chromosomal reorganizations between species. Additionally, we observe that some of the verified species-specific inversions are split across multiple genomic regions when mapped against a heterospecific reference. CONCLUSIONS: Inaccurate identification of chromosomal rearrangements as well as biased population genetic measures could potentially lead to erroneous interpretation of species-specific genomic diversity, impede the resolution of local adaptation, and thus, impact predictions of their genomic potential to respond to climatic and other environmental perturbations.

Animals↗

PubMed1982

Physical and genetic analysis of deletion mutants of plasmid R91-5 and the cloning of transfer genes in Pseudomonas aeruginosa.

We isolated deletion mutants of Pseudomonas aeruginosa plasmid R91-5 by both in vitro and in vivo means. Many of the deletion mutants selected on the basis of resistance to donor-specific phages fell into a few groups of apparently identical mutants, although the mutants were nonsibs. By analyzing plasmids with large deletions, we found that the essential replication genes of R91-5 were within a 3.85-kilobase region between coordinates 45.5 and 48.9. The origin of plasmid transfer (oriT) was mapped to a 4.5-kilobase region between coordinates 1.7 and 6.2. We indirectly determined the direction of plasmid transfer from oriT. By combining the data from our analysis of the deletions with data from complementation tests between cloned R91-5 fragments and known reference mutants, we ordered and mapped the 10 known transfer (tra) cistrons of R91-5. All of the tra cistrons mapped within the Tra2 region, and their order was as follows: traX, -Y, -T, -Q, -(V, R), -U, -(S, Z), -W (the cistrons in parentheses could not be ordered with respect to each other).

Cloning, Molecular↗

PubMed1990

High-performance tryptic mapping of recombinant bovine somatotropin.

Experiments are described that have lead to the development of a highly reproducible tryptic map of recombinant DNA derived bovine somatotropin (rbSt). Tryptic digestion of rbSt at 37 degrees C results in the formation of a precipitate. Preliminary characterization of the precipitate suggests that its formation is due to the association of intermediate tryptic fragments. An examination of the temperature dependence of the digestion has revealed that precipitate formation is inhibited when digestion is performed at 10 degrees C or less. The combination of a 5-mg sample, the use of highly purified trypsin, and digestion at 5 degrees C generate a tryptic map that exhibits an average 1.3% RSD (0.5-3.6%) for all anticipated fragments. Validation studies demonstrate that while the peak response precision is rugged to daily variation of operators or chromatographic systems, the fragment retention is not. This dictates that peaks be assigned by qualitative pattern recognition. Assay ruggedness in the peak response domain allows for the implementation of quantitative methods for the comparison of rbSt reference standard and sample tryptic maps. The assay is linear for all anticipated fragments within 50-150% of the operating range. Specificity is established by assay of pituitary somatotropins from other species and rbSt analogs produced by site-specific mutagenesis. The data demonstrate that all single amino acid substitutions examined are identified by using the technique. Assay sensitivity is validated for selected tryptic fragments through analysis of reference standard digests spiked with known amounts of rbSt analog digests. The data indicate that potential impurities of 3.2, 2.0, and 4.5% can be quantitated with statistical confidence in the tryptic fragments T1, T10, and T23 + 25, respectively.

Amino Acid Sequence↗

PubMed1989

Molecular and cellular studies of tryptophanyl-tRNA synthetase using monoclonal antibodies. Evaluation of a common antigenic determinant in eukaryotic, prokaryotic and archaebacterial enzymes which maps outside the catalytic domain.

Monoclonal antibodies referred to as Am1, Am2 and Am3 against highly purified bovine tryptophanyl-tRNA synthetase were prepared. Am2 antibodies inhibit the Trp-tRNA synthetase activity and interact with the active truncated enzyme forms (dimers of either 40-kDa or 51-kDa fragments) produced by limited proteolysis. Am1 and Am3 antibodies exert no effect on the Trp-tRNA synthetase activity; epitopes recognized by them are mapped close to one another and reside at the dispensable part of the Trp-tRNA synthetase molecule. Am1 cross-reacts with Trp-tRNA synthetases of eukaryotic, prokaryotic and archaebacterial species, as revealed by immunoblot analysis. A rapid two-step technique was developed for isolating electrophoretically homogeneous Trp-tRNA synthetase from Escherichia coli. The purified enzyme interacted with Am1, but not with Am2 and Am3 antibodies taken at the same concentrations. As in the case of eukaryotic Trp-tRNA synthetase, Am1 did not influence the activity of Trp-tRNA synthetase from E. coli. From the aforementioned results it follows that: (a) the conservation of part of the Trp-tRNA synthetase structure which is not directly involved in the formation of the catalytic centre of prokaryotic and eukaryotic Trp-tRNA synthetases suggests that the dispensable part of the molecule might be involved in some additional biological function(s) of Trp-tRNA synthetase besides tRNA(Trp) charging; (b) the common antigenic determinant in Trp-tRNA synthetase of eukaryotes, prokaryotes and archaebacteria indicates that this enzyme was presumably present in the common ancestor of the above organisms.

Amino Acyl-tRNA Synthetases↗

PubMed1984

EEG assessment of brain activity: spatial aspects, segmentation and imaging.

High temporal resolution and sensitivity to index different functional brain states makes the EEG a powerful tool in psychophysiology. Its full potential can now be utilized since recording technology and computational power for the large data masses has become affordable. However, basic traditional strategies in EEG need reviewing. Conventional, spontaneous or evoked EEG traces which are used for various complex analyses give ambiguous information on EEG power (amplitude) and phase for a given point on the scalp. Principally, analysis should first be done over space, then over time, to avoid ambiguities or pre-selections. First or second spatial derivative computations can provide "reference-free" data for analyses over time. We propose to use direct, spatial approaches for the analysis of the scalp EEG field distributions when simultaneous recordings in several EEG channels can be examined. The ambiguity of the conventional EEG waveshapes results in different, equally "correct" scalp maps of EEG power of the same multichannel data for different reference electrodes. An exception are scalp maps of EEG power computed against the common, average reference, as they are related to the reference-free spatial distribution (maps) of the maximal and minimal (extreme) field values over time, and thus are directly interpretable in terms of net orientation of the generator process. A proposed, reference-free EEG segmentation into epochs of periodically stationary spatial distributions of the mapped scalp EEG fields uses the locations of maximal and minimal (extreme) field values at each moment in time as classifiers, and thus avoids the priviledging of two arbitrarily chosen recording points in the field.

Brain↗