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PubMed1999

Model-based morphological segmentation and labeling of coronary angiograms.

A method for extraction and labeling of the coronary arterial tree (CAT) using minimal user supervision in single-view angiograms is proposed. The CAT structural description (skeleton and borders) is produced, along with quantitative information for the artery dimensions and assignment of coded labels, based on a given coronary artery model represented by a graph. The stages of the method are: 1) CAT tracking and detection; 2) artery skeleton and border estimation; 3) feature graph creation; and iv) artery labeling by graph matching. The approximate CAT centerline and borders are extracted by recursive tracking based on circular template analysis. The accurate skeleton and borders of each CAT segment are computed, based on morphological homotopy modification and watershed transform. The approximate centerline and borders are used for constructing the artery segment enclosing area (ASEA), where the defined skeleton and border curves are considered as markers. Using the marked ASEA, an artery gradient image is constructed where all the ASEA pixels (except the skeleton ones) are assigned the gradient magnitude of the original image. The artery gradient image markers are imposed as its unique regional minima by the homotopy modification method, the watershed transform is used for extracting the artery segment borders, and the feature graph is updated. Finally, given the created feature graph and the known model graph, a graph matching algorithm assigns the appropriate labels to the extracted CAT using weighted maximal cliques on the association graph corresponding to the two given graphs. Experimental results using clinical digitized coronary angiograms are presented.

Algorithms↗

PubMedDate not supplied

Complete nucleotide sequence of Japanese flounder (Paralichthys olivaceus) mitochondrial genome: structural properties and cue for resolving teleostean relationships.

We cloned and sequenced the complete mitochondrial genome of Japanese flounder (Paralichthys olivaceus). A circular 17,090 bp mitochondrial genome from the flounder contains 37 structural genes as in other vertebrates so far reported. This is the first report of the complete mitochondrial sequence from a higher teleostean fish (Acanthopterygii). The organization including gene order is quite similar to that of other teleostean fishes as well as placental mammals. The putative control region of the Japanese flounder mitochondrial genome contains a length variable region of about a 74 bp tandem repeat cluster. As a preliminary study we adopted the maximum likelihood and neighbor-joining inference methods to examine phylogenetic relationships among teleostean and related fishes. Comparisons of amino acid sequences of protein-coding genes and nucleotide sequences of tRNA genes resolved some middle to deep branches among some teleostean fishes. The flounder mitochondrial genome does not show an indication of evolutionary rate difference among teleosts leading to difficulty in phylogenetic analyses, and our data is useful for future evolutionary studies dealing with higher teleostean fishes.

Animals↗

PubMed1999

Structure of the trp RNA-binding attenuation protein, TRAP, bound to RNA.

The trp RNA-binding attenuation protein (TRAP) regulates expression of the tryptophan biosynthetic genes of several bacilli by binding single-stranded RNA. The binding sequence is composed of eleven triplet repeats, predominantly GAG, separated by two or three non-conserved nucleotides. Here we present the crystal structure of a complex of TRAP and a 53-base single-stranded RNA containing eleven GAG triplets, revealing that each triplet is accommodated in a binding pocket formed by beta-strands. In the complex, the RNA has an extended structure without any base-pairing and binds to the protein mostly by specific protein-base interactions. Eleven binding pockets on the circular TRAP 11-mer form a belt with a diameter of about 80 A. This simple but elegant mechanism of arresting the RNA segment by encircling it around a protein disk is applicable to both transcription, when TRAP binds the nascent RNA, and to translation, when TRAP binds the same sequence within a non-coding leader region of the messenger RNA.

Bacterial Proteins↗

PubMed1995

Expression of Sry, the mouse sex determining gene.

In the mouse, Sry is expressed by germ cells in the adult testis and by somatic cells in the genital ridge. Transcripts in the former exist as circular RNA molecules of 1.23 kb, which are unlikely to be efficiently translated. We have used RNase protection to map the extent of the less abundant Sry transcript in the developing gonad. We demonstrate that it is a linear mRNA derived from a single exon. This begins in the unique region 5' of the protein coding region and extends several kilobases into the 3' arm of the large inverted repeat which bounds the Sry genomic locus. Knowledge of this transcript, which is very different from that of the human SRY gene, allows us to predict its protein product and reveals several features which may be involved in translational control. Our data is also consistent with there being two promoters for the Sry gene, a proximal one that gives functional transcripts in the genital ridge and a distal promoter used in germ cells in the adult testis. As RNase protection is a quantitative technique, a detailed timecourse of Sry expression was carried out using accurately staged samples. Sry transcripts are first detectable just after 10.5 days post coitum, they reach a peak at 11.5 days and then decline sharply so that none are detected 24 hours later. This was compared with anti-Müllerian hormone gene expression, an early marker of Sertoli cells and the first known downstream gene of Sry. Amh expression begins 20 hours after the onset of Sry expression at a time when Sry transcripts are at their peak. While this result does not prove a direct interaction between the two genes, it defines the critical period during which Sry must act to initiate Sertoli cell differentiation.

Amino Acid Sequence↗

PubMed1992

Projections and chemical coding of neurons with immunoreactivity for nitric oxide synthase in the guinea-pig small intestine.

The distribution of nitric oxide synthase (NOS) immunoreactivity was investigated in the guinea-pig small intestine. There were many immunoreactive nerve cell bodies in the myenteric plexus but very few in submucous ganglia. NOS immunoreactivity was not found in non-neuronal cells except for rare mucosal endocrine cells. Abundant immunoreactive nerve fibres in both myenteric and submucous ganglia, and in the circular muscle, arose from myenteric nerve cells whose axons projected anally along the intestine. NOS immunoreactivity coexisted with VIP-immunoreactivity, but not with substance P immunoreactivity. We conclude that nitric oxide synthase is located in a sub-population of enteric neurons, amongst which are inhibitory motor neurons that supply the circular muscle layer.

Amino Acid Oxidoreductases↗

PubMed1970

Coding and conformational properties of oligonucleotides modified with the carcinogen N-2-acetylaminofluorene.

The present studies were undertaken to determine the mechanism by which attachment of the carcinogen N-2-acetylaminofluorene to guanosine residues in nucleic acids distors their structure and function. Oligonucleotides were modified with N-acetoxy-2-acetylaminofluorene, repurified, and their base compositions analyzed. Evidence is presented that acetylaminofluorene residues bound to guanosines in GpUpU, ApApG, or poly (U,G) inactivates their function in codon recognition. Circular dichroism spectra suggest that this is caused by gross conformational changes in these compounds involving both a rotation about the glycosidic bond of guanosine residues bearing N-2-acetylaminofluorene, as well as stacking interactions between the drug and bases adjacent to the substituted guanosine.

Acetates↗

PubMed1988

The DNA intermediate in yeast Ty1 element transposition copurifies with virus-like particles: cell-free Ty1 transposition.

Yeast Ty1 elements are retrotransposons that transpose via an RNA intermediate found in a virus-like particle (Ty-VLP). A Ty-encoded reverse transcriptase activity found inside the particles is capable of giving rise to full-length reverse transcripts. The predominant form of these reverse transcripts is a full-length linear duplex DNA. We have developed a cell-free system for transposition of Ty1 DNA molecules into a bacteriophage lambda target. Purified Ty-VLPs and target DNA are the only macromolecular components required for the transposition reaction. A TYB-encoded protein, p90-TYB, contains amino acid sequences that are similar to those of retroviral integrase proteins. Mutations in the integrase coding region abolish transposition both in vivo and in vitro.

Antibodies, Monoclonal↗

PubMed1977

New mini-ColE1 as a molecular cloning vehicle.

A new mini-ColE1 plasmid, designated pAC105, was isolated. It has a molecular weight of 1.6 X 10(6) and carries information for its self-replication as well as information for conferring colicin E1 immunity upon its host. Furthermore, pAC105 undergoes replication in the presence of chloramphenicol even when a foreign deoxyribonucleic acid (pSC101) is inserted into its single EcoRI restriction site. Studies in minicell-producing strains demonstrate that pAC105 codes for only two or three polypeptides of low molecular weight. The advantages of using it as a molecular cloning vehicle are discussed.

Bacterial Proteins↗

PubMed2002

Numerical computation of the scattering matrix of an electromagnetic resonator.

A method is presented to investigate diffraction of an electromagnetic plane wave by an infinitely thin infinitely conducting circular cylinder with longitudinal slots. It is based on the use of the combined boundary conditions method that consists of expressing the continuity of the tangential components of both the electric and the magnetic fields in a single equation. This method proves to be very efficient for this kind of problem and leads to fast numerical codes. The scattering matrix that is obtained from this theory can then be used in a multiscattering method to study wave propagation in square arrays of such resonators with an emphasis on the low-frequency behavior.

Journal Article↗

PubMed1997

Neuronal encoding of sound direction in the auditory midbrain of the rainbow trout.

Acoustical stimulation causes displacement of the sensory hair cells relative to the otoliths of the fish inner ear. The swimbladder, transforming the acoustical pressure component into displacement, also contributes to the displacement of the hair cells. Together, this (generally) yields elliptical displacement orbits. Alternative mechanisms of fish directional hearing are proposed by the phase model, which requires a temporal neuronal code, and by the orbit model, which requires a spike density code. We investigated whether the directional selective response of auditory neurons in the midbrain torus semicircularis (TS; homologous to the inferior colliculus) is based on spike density and/or temporal encoding. Rainbow trout were mounted on top of a vibrating table that was driven in the horizontal plane to simulate sound source direction. Rectilinear and elliptical (or circular) motion was applied at 172 Hz. Generally, responses to rectilinear and elliptical/circular stimuli (irrespective of direction of revolution) were the same. The response of auditory neurons was either directionally selective (DS units, n = 85) or not (non-DS units, n = 106). The average spontaneous discharge rate of DS units was less than that of non-DS units. Most DS units (70%) had spontaneous activities < 1 spike per second. Response latencies (mode at 18 ms) were similar for both types of units. The response of DS units is transient (19%), sustained (34%), or mixed (47%). The response of 75% of the DS units synchronized to stimulus frequency, whereas just 23% of the non-DS responses did. Synchronized responses were measured at stimulus amplitudes as low as 0.5 nm (at 172 Hz), which is much lower than for auditory neurons in the medulla of the trout, suggesting strong convergence of VIIIth nerve input. The instant of firing of 42% of the units was independent of stimulus direction (shift <15 degrees), but for the other units, a direction dependent phase shift was observed. In the medial TS spatial tuning of DS units is in the rostrocaudal direction, whereas in the lateral TS all preferred directions are present. On average, medial DS units have a broader directional selectivity range, are less often synchronized, and show a smaller shift of the instant of firing as a function of stimulus direction than lateral DS units. DS response characteristics are discussed in relation to different hypotheses. We conclude that the results are more in favor of the phase model.

Air Sacs↗

PubMed1997

Expression of wild-type and modified proalpha chains of human type I procollagen in insect cells leads to the formation of stable [alpha1(I)]2alpha2(I) collagen heterotrimers and [alpha1(I)]3 homotrimers but not [alpha2(I)]3 homotrimers.

Insect cells coinfected with a baculovirus coding for the proalpha1(I) chain of human type I procollagen and a double promoter virus coding for the alpha and beta subunits of human prolyl 4-hydroxylase produced homotrimeric [proalpha1(I)]3 procollagen molecules. The use of an additional virus coding for the proalpha2(I) chain led to the formation of a heterotrimeric molecule with the correct 2:1 ratio of proalpha1 to proalpha2 chains of type I procollagen (proalpha1(I) and proalpha2(I) chains, respectively), unless the proalpha1(I) chain was expressed in a relatively large excess. Replacement of the sequences coding for the signal peptide and the N propeptide of the proalpha1(I) chain with those of the proalpha1(III) chain increased level of expression of the proalpha1(I) chain, whereas no similar effect was found when the corresponding modification was made to the virus coding for the proalpha2(I) chain. Molecules containing such modified N propeptides were found to be processed at their N terminus more rapidly than those containing the wild-type propeptides. The Tm of the type I collagen homotrimer was similar to that of the heterotrimer, both values being about 42-43 degrees C when determined by circular dichroism. The wild-type proalpha2(I) chain formed no homotrimers. Replacement of the C propeptide of the proalpha2(I) chain with that of the proalpha1(I) chain or proalpha1 chain of type III procollagen (proalpha1(III) chain) led to the formation of homotrimers, but the alpha2(I) chains in such molecules were completely digested by pepsin in 1 h at 22 degrees C. The data thus suggest that, in addition to control at the level of the C propeptide, other restrictions may exist at the level of the collagen domain that prevent the formation of stable homotrimeric [proalpha2(I)]3 molecules in insect cells.

Animals↗

PubMed1983

Formation of genes coding for hybrid proteins by recombination between related, cloned genes in E. coli.

We describe a method for the formation of hybrid genes by in vivo recombination between two genes with partial sequence homology. DNA structures consisting of plasmid vector sequences, flanked by the alpha 2 interferon gene on the one side and a portion of the alpha 1 interferon gene (homology about 80%) on the other, were transfected into E. coli SK1592. Appropriate resistance markers allowed the isolation of colonies containing circular plasmids which arose by in vivo recombination between the partly homologous interferon gene sequences. Eleven different recombinant genes were identified, six of which encoded new hybrid interferons not easily accessible by recombinant DNA techniques.

Amino Acid Sequence↗

PubMed2026

Development of a cell-based nanoluciferase reporter system for high-throughput screening of HBV cccDNA inhibitors.

Hepatitis B virus (HBV) persistence is sustained by the viral covalently closed circular DNA (cccDNA) minichromosome, which remains a major barrier to curative antiviral therapies. The lack of reliable quantitative cccDNA detection methods and surrogate markers has hindered efforts to target cccDNA in antiviral high-throughput screening (HTS). Here, we established a novel inducible cccDNA-dependent nanoluciferase (NLuc) reporter cell line, designated HepBLE12, by inserting an in-frame 11-amino acid split-NLuc HiBiT tag into the precore (pC) coding region of an HBV transgene. The resulting 1.3-kDa HiBiT tag on pC serves as the detection module of the split NLuc system, generating quantitative luminescence upon high-affinity complementation with the cognate 18-kDa LgBiT subunit in cell lysates. Notably, the HiBiT assay enables direct detection of intracellular HiBiT-pC protein rather than secreted HBeAg, providing a reporter signal more closely linked to cccDNA activity. HepBLE12 cells exhibited inducible and robust viral DNA replication, and the cccDNA-dependent HiBiT signal was validated under diverse experimental conditions that modulate cccDNA formation or transcription. We further miniaturized the assay to a 384-well format and optimized key parameters following standard HTS assay development practices. The assay was successfully automated and demonstrated excellent performance in a multi-day variability study and a pilot screen, with signal-to-background (S/B) ≈ 9, coefficient of variance (CV) < 10%, and average Z-factor value of 0.74, exceeding canonical HTS quality benchmarks. Together, the HepBLE12 cell-based HTS platform provides a robust and practical tool for identifying inhibitors targeting HBV cccDNA.

Hepatitis B virus↗

PubMed2002

Detection of defects in cylindrical structures using a time reverse method and a finite-difference approach.

The detection and characterization of defects in structures is an important issue in non-destructive testing. To avoid the scanning of large samples, guided elastic waves, which propagate along the structure, are excited. These waves interact with a defect, which results in a scattered wave field. In an experiment, the displacements of these scattered waves are recorded over time for a fixed axial coordinate at a number of circumferential positions of a circular cylindrical tube. Since in complex structures it is difficult to determine the axial and particularly circumferential position of the defect directly from the time signals, a time reversed numerical simulation is performed. There the measured displacement histories are reversed in time and used as displacement excitations in a simulation of the tested structure. A three-dimensional code in cylindrical coordinates, based on a velocity-stress finite-difference method, is used to simulate the wave propagation. As long as the geometric and material parameters are chosen equivalent to the performed experiment, the scattered waves travel back through the simulated structure and interfere, even if no defect is present in the numerical model. The result is an increase of the amplitudes of the stress and displacement components at the location where the defect was in the tested sample.

Journal Article↗

PubMed1984

Interallelic complementation in an inborn error of metabolism: genetic heterogeneity in argininosuccinate lyase deficiency.

We used complementation analysis as a probe for the detection of genetic heterogeneity within a single locus affected in a human disease, argininosuccinate lyase (L-argininosuccinate arginine-lyase, EC 4.3.2.1) deficiency. Fibroblasts cultured from 28 unrelated patients were fused in all possible pairwise combinations, and the argininosuccinate lyase activity in heterokaryons was assayed by measuring the incorporation of 14C from L-[ureido-14C]citrulline into acid-precipitable material. Partial complementation was observed in fusions involving 20 of the 28 strains, with the lyase activity increasing from 2- to 10-fold. Thirteen of the mutants were identified by the complementation analysis as being phenotypically unique. Of the 20 complementing strains, 3 were remarkable because they participated in all but 2 of the 32 positive complementation tests; 2 others constituted a unique subgroup that produced the highest increases in argininosuccinate lyase activity of all fusions. The 8 strains that did not complement any others consisted of two types: 3 mutants with the highest residual argininosuccinate lyase activity of all strains and 5 mutants with low residual activity. All of the mutants mapped to a single major complementation group. The data could be summarized as a circular complementation map with an attached linear tail, the mutants being distributed among 12 subgroups in a complex pattern. We conclude that all of these mutants are affected at a single locus, that extensive genetic heterogeneity is present in the mutant population, and that the affected locus in argininosuccinate lyase deficiency is likely to be the structural gene coding for that enzyme.

Alleles↗

PubMed1990

Delta hepatitis. The disease and the virus.

Delta hepatitis virus is a new human pathogen always found associated with hepatitis B virus (HBV) causing both fulminant hepatitis and the accelerated progression of pre-existing HBV hepatitis. The virus is coated in HBsAg and contains circular single stranded RNA genome with very high intramolecular base-pairing, similar to the genomes of plant viroids, and the HDV antigen, a specific marker of HDV infection also found in the nuclei of infected hepatocytes. The genome is about 1700 nucleotides long and of minus polarity with a major constant antigenomic Open Reading Frame (ORF) which codes for the antigen. Replication of viral RNA appears to proceed by a rolling circle mechanism and specific self-cleavage and self-ligation of both genomic and antigenomic HDV RNA strands has been demonstrated in vitro. HDV appears to require HBsAg for its propagation and hepatotropism but not to depend on HBV for replication.

Animals↗

PubMed2026

The complete Chloroplast Genome of Dianthus Helenae, an Endemic Species with Medicinal Potential from the Nuratau Mountains, Uzbekistan.

Dianthus helenae Vved. is an endemic medicinal species of the Nuratau Mountains, Uzbekistan, and its genomic resources have remained largely unavailable. In this study, we sequenced, assembled, and characterized the complete chloroplast genome of D. helenae and evaluated its phylogenetic position within Dianthus. The plastome exhibited a typical circular quadripartite structure with a total length of 149,567 bp, comprising a large single-copy (LSC) region of 82,856 bp, a small single-copy (SSC) region of 17,105 bp, and a pair of inverted repeats (IRs) of 24,803 bp each. The genome contained the typical set of chloroplast genes, including protein-coding genes, transfer RNAs, and ribosomal RNAs, with duplicated genes located in the IR regions. Phylogenetic analysis based on complete chloroplast genome sequences strongly supported the placement of D. helenae within Dianthus and recovered it as a distinct lineage relative to other sampled species. Sliding window analysis of nucleotide diversity revealed uneven sequence variation across the plastome, with higher variability in the SSC and LSC regions than in the IRs. Several highly variable loci, including trnK-UUU , rps16-trnQ-UUG , rpl32, ycf1, and ndh-associated regions, were identified as potential molecular markers. These results provide an important genomic resource for Dianthus and establish a foundation for future phylogenetic, taxonomic, conservation, and molecular identification studies of this endemic Central Asian species.

Genome, Chloroplast↗

PubMed1992

V(D)J recombination: broken DNA molecules with covalently sealed (hairpin) coding ends in scid mouse thymocytes.

Lymphoid cells from scid mice initiate V(D)J recombination normally but have a severely reduced ability to join coding segments. Thymocytes from scid mice contain broken DNA molecules at the TCR delta locus that have coding ends, as well as molecules with signal ends, whereas in normal mice we previously detected only signal ends. Remarkably, these coding (but not signal) ends are sealed into hairpin structures. The formation of hairpins at coding ends may be a universal, early step in V(D)J recombination; this would provide a simple explanation for the origin of P nucleotides in coding joints. These findings may shed light on the mechanism of cleavage and suggest a possible role for the scid factor.

Animals↗