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At least 1,495 records · Page 83Linked to original sources
PubMed1999

Similarity measurement method for the classification of architecturally differentiated images.

A similarity measurement method for the classification of architecturally differentiated image sections is described. The strength of the method is demonstrated by performing the complex task of assigning severity grading (Gleason grading) to histological slides of prostate cancer. As shown, all that is required to employ the method is a small set of preclassified images. The images can be real world images acquired by means of a camera, computer tomography, etc., or schematic drawings representing samples of different classes. The schematic option allows a quick test of the method for a particular classification problem.

Computers↗
PubMed2001

The LAX1 and FRIZZY PANICLE 2 genes determine the inflorescence architecture of rice by controlling rachis-branch and spikelet development.

We have analyzed two mutants that exhibit altered panicle architecture in rice (Oryza sativa L.). In lax1-2, which is a new and stronger allele of the previously reported lax mutant, initiation and/or maintenance of rachis-branches, lateral spikelets, and terminal spikelets was severely prevented. In situ hybridization analysis using OSH1, a rice knotted1 (kn1) ortholog, confirmed the absence of lateral meristems in lax1-2 panicles. These defects indicate that the LAX1 gene is required for the initiation/maintenance of axillary meristems in the rice panicle. In addition to its role in forming lateral meristems, the wild-type LAX1 gene acts as a floral meristem identity gene which specifies the terminal spikelet meristem. A comparison of the defects in lax1-1 and lax1-2 plants suggested that the sensitivities to reduced LAX1 activity were not uniform among different types of meristems. In the fzp2 mutant panicle, the basic branching pattern of the panicle was indistinguishable from that of the wild type; however, specification of both terminal and lateral spikelet meristems was blocked, and sequential rounds of branching occurred at the point where the spikelet meristems are initiated in the wild-type panicle. This resulted in the generation of a panicle composed of excessive ramification of rachis-branches. The lax1-1 fzp2 double mutants exhibited a novel, basically additive, phenotype, which suggests that LAX1 and FZP2 function in genetically independent pathways.

Alleles↗
PubMed1996

Dynamic changes in nuclear architecture during mitosis: on the role of protein phosphorylation in spindle assembly and chromosome segregation.

During mitosis, the vertebrate cell nucleus undergoes profound changes in architecture. At the onset of mitosis, the nuclear envelope breaks down, the nuclear lamina is depolymerized, and interphase chromatin is condensed to chromosomes. Concomitantly, cytoplasmic microtubules are reorganized into a mitotic spindle apparatus, a highly dynamic structure required for the segregation of sister chromatids. Many of the above events are controlled by reversible phosphorylation. Hence, our laboratory is interested in characterizing the kinases involved in promoting progression through mitosis and in identifying their relevant substrates. Prominent among the kinases responsible for regulating entry into mitosis is the Cdc2 kinase, the first member of the cyclin dependent kinase (Cdk) family. Recently, we found that Cdc2 phosphorylates HsEg5, a human kinesin-related motor protein associated with centrosomes and the spindle apparatus. Our results indicate that phosphorylation regulates the association of HsEg5 with the mitotic spindle and that the function of this plus-end directed motor is essential for centrosome separation and bipolar spindle formation. Another kinase implicated in regulating progression through mitosis is Plk1 (polo-like kinase 1), the human homologue of the Drosophila gene product "polo." By antibody microinjection we have found that Plk1 is required for the functional maturation of centrosomes and hence for entry into mitosis. Furthermore, we found that microinjected anti-Plk1 antibodies caused a more severe block to cell cycle progression in diploid fibroblasts than in immortalized tumor cells. This observation hints at the existence of a checkpoint linking Cdc2 activation to the presence of functional centrosomes.

Animals↗
PubMed2002

Spatial preservation of nuclear chromatin architecture during three-dimensional fluorescence in situ hybridization (3D-FISH).

3D-FISH has become a major tool for studying the higher order chromatin organization in the cell nucleus. It is not clear, however, to what extent chromatin arrangement in the nucleus after fixation and 3D-FISH still reflects the order in living cells. To study this question, we compared higher order chromatin arrangements in living cells with those found after the 3D-FISH procedure. For in vivo studies we employed replication labeling of DNA with Cy3-conjugated nucleotides and/or chromatin labeling by GFP-tagged histone 2B. At the light microscope level, we compared the intranuclear distribution of H2B-GFP-tagged chromatin and the positions of replication-labeled chromatin domains in the same individual cells in vivo, after fixation with 4% paraformaldehyde, and after 3D-FISH. Light microscope data demonstrate a high degree of preservation of the spatial arrangement of approximately 1-Mb chromatin domains. Subsequent electron microscope investigations of chromatin structure showed strong alterations in the ultrastructure of the nucleus caused mainly by the heat denaturation step. Through this step chromatin acquires the appearance of a net with mesh size of 50-200 nm roughly corresponding to the average displacement of the chromatin domains observed at light microscope level. We conclude that 3D-FISH is a useful tool to study chromosome territory structure and arrangements down to the level of approximately 1-Mb chromatin domain positions. However, important ultrastructural details of the chromatin architecture are destroyed by the heat denaturation step, thus putting a limit to the usefulness of 3D-FISH analyses at nanometer scales.

Carbocyanines↗
PubMed2001

Leishmania donovani: evolution and architecture of the splenic cellular immune response related to control of infection.

Infection with the protozoan Leishmania donovani in humans is usually subclinical. Parasites probably persist for the life of the host and the low-level infection is controlled by the cellular immune response. To better understand the mechanisms related to the control of infection, we studied the evolution and architecture of the splenic cellular immune response in a murine model that is most representative of human subclinical infection. Following systemic inoculation with L. donovani, the parasites were primarily localized to the macrophage-rich splenic red pulp. There was an initial increase in the numbers of T cells and dendritic cells in the periarteriolar lymphoid sheath and marginal zone, but the red pulp (where parasitized macrophages were prominent) remained free of these cells until later in the course of infection. Thus, T cells did not colocalize with parasitized red pulp macrophages until later in the course of infection. Early in the course of infection, IL-10 production within the marginal zone and TGF-beta production by cells in the red pulp were prominent. These macrophage-inhibitory cytokines may contribute to the establishment of the infection and early parasite replication. By day 28 of infection, when the visceral parasite burden began to decline, the number of IL-10-producing spleen cells was back to the baseline level, but IFN-gamma production was higher and the number of IL-12-producing cells was increased dramatically. At this time T cells and dendritic cells had moved out of the lymphoid follicle and marginal zone into the red pulp where the parasites were located. These findings therefore suggest that control of infection is associated with IFN-gamma and IL-12 production and migration of T cells and dendritic cells to the site of chronic parasitism.

Animals↗
PubMed2001

Tetrakis(octadecylthio)Tetrathiafulvalene: Influence of Long Alkyl Chains on the Architecture and Electrochemical Properties of LB Films.

We present here a study of a tetrathiafulvalene derivative, tetrakis(octadecylthio)tetrathiafulvalene (TTFH), arranged in LB films together with a detailed characterization process by means of UV-vis, IR, SEM, and X-ray diffraction that has allowed us to propose a packing model. These films were exposed to iodine vapor and this doping process was carefully followed using UV-vis and IR spectroscopy. The redox properties of the TTFH were studied in both organic solution and LB films. The results have been interpreted in terms of the molecular structure and the LB film architecture. Copyright 2001 Academic Press.

Journal Article↗
PubMed1999

Hip bone trabecular architecture shows uniquely distinctive locomotor behaviour in South African australopithecines.

Cancellous bone retains structural and behavioural properties which are time and strain-rate dependent. As the orientation of the trabeculae (trajectories) follows the direction of the principal strains imposed by daily loadings, habitual postural and locomotor behaviours are responsible for a variety of trabecular architectures and site-specific textural arrangements of the pelvic cancellous network. With respect to the great ape condition, the human trabecular pattern is characterized by a distinctive ilioischial bundle, an undivided sacropubic bundle, and a full diagonal crossing (approximately 100 degrees) over the acetabulum between the ilioischial and the sacropubic bundles. Advanced digital image processing (DIP) of hip bone radiographs has revealed that adolescent and adult South African australopithecines retained an incompletely developed human-like trabecular pattern associated with gait-related features that are unique among the extant primates.

Adolescent↗
PubMed1993

Three-dimensional architecture of human alpha 2-macroglobulin transformed with methylamine.

A frozen-hydrated sample embedded in vitreous ice of human alpha 2-macroglobulin transformed by methylamine was imaged by cryoelectron microscopy and reconstructed in three dimensions. In the reconstruction, the cage-like architecture of this protease inhibitor is fully revealed with a clear visualization of two lozenge-shaped lateral walls connected by thin bridges. The shape and dimensions of the internal cavity normally containing the trapped protease(s) is described. The possible locations of the thiol ester sites and inter-subunit connections are also discussed.

Image Processing, Computer-Assisted↗
PubMed1995

Towards the molecular architecture of the asymmetric unit membrane of the mammalian urinary bladder epithelium: a closed "twisted ribbon" structure.

The asymmetric unit membrane (AUM) forms numerous plaques covering the apical surface of mammalian urinary bladder epithelium. These plaques contain four major integral membrane proteins called uroplakins Ia, Ib, II and III, which form particles arranged in a well-ordered hexagonal lattice with p6 symmetry and a lattice constant of 16.5 nm. Bovine AUM plaques negatively stained with anionic sodium silicotungstate revealed structural detail to 3.1 nm resolution. Correlation averaging resolved each particle into 12 stain-excluding domains arranged in two concentric rings (inner ring radius (rm) = 3.7 nm, outer ring radius (rout) = 6.6 nm), each with six domains which were rotated by roughly 30 degrees relative to each other. Negative staining with cationic uranyl formate increased the resolution to 2.2 nm and unveiled distinct connections between adjacent AUM particles. These connections may provide a molecular basis for the observed insolubility of the plaques in many detergents. Examination of the luminal face of freeze-dried/unidirectionally metal-shadowed AUM plaques established a left-handed vorticity of the 16 nm protein particles, whereas the cytoplasmic face exhibited no significant surface corrugations. Three-dimensional reconstruction from sodium silicotungstate-stained specimens revealed the AUM particles to be built of six "V-shaped" subunits anchored upright in the membrane. The mass density distribution within uranyl formate-stained AUM particles was similar except that the inner tip of each V was bridged to the outer tip of an adjacent V, so that the 16 nm AUM particle appeared as a continuous, "twisted ribbon" embracing a central cavity. Finally, mass measurements of unstained/freeze-dried plaques by scanning transmission electron microscopy yielded a total mass of 1,120 kDa per membrane-bound AUM particle. By imposing constraints on the possible uroplakin stoichiometries within AUM plaques, these data provide a first glimpse of the molecular architecture of the 16 nm particles constituting the plaques.

Animals↗
PubMed1996

Enzymatic formation of modified nucleosides in tRNA: dependence on tRNA architecture.

Information is still quite limited concerning the structural requirements in tRNA molecules for their post-transcriptional maturation by base and ribose modification enzymes. To address this question, we have chosen as the model system yeast tRNAAsp that has a known three-dimensional structure and the in vivo modifying machinery of the Xenopus laevis oocyte able to act on microinjected tRNA precursors. We have systematically compared the modification pattern of wild-type tRNAAsp with that of a series of structural mutants (21 altogether) altered at single or multiple positions in the D-, T-and the anticodon branch, as well as in the variable region. The experimental system allowed us to analyze the effects of structural perturbations in tRNA on the enzymatic formation of modified nucleosides at 12 locations scattered over the tRNA cloverleaf. We found that the formation of m1G37 and psi 40 in the anticodon loop and stem and psi 13 in the D-stem, were extremely sensitive to 3D perturbations. In contrast, the formation of T54, psi 55 and m1A58 in the T-loop, m5C49 in the T-stem and m2G6 in the amino acid accepting stem were essentially insensitive to change in the overall tRNA architecture; these modified nucleosides were also formed in appropriate minimalist (stems and loops) tRNA domains. The formation of m2G26 at the junction between the anticodon and the D-stem, of Q34 and manQ34 in the anticodon loop were sensitive only to drastic structural perturbation of the tRNA. Altogether, these results reflect the existence of different modes of tRNA recognition by the many different modifying enzymes. A classification of this family of maturation enzymes into two major groups, according to their sensitivities to structural perturbations in tRNA, is proposed.

Animals↗
PubMed1997

The 2.4 A resolution crystal structure of boar seminal plasma PSP-I/PSP-II: a zona pellucida-binding glycoprotein heterodimer of the spermadhesin family built by a CUB domain architecture.

The crystal structure of porcine seminal plasma spermadhesin PSP-I/PSP-II heterodimer has been determined in two crystal forms by multiple isomorphous replacement in an hexagonal crystal (space group P6(1)22) and molecular replacement in a trigonal crystal of space group P3(2)21. The crystal structure has been refined at 2.4 A resolution to an R-factor of 20.0% (Rfree = 25.9%) for 14,809 independent reflections with intensities greater than 2 sigma (I), with root-mean-square deviations of 0.009 A and 1.657 degrees from ideal bond lengths and bond angles, respectively. The final model includes 1688 non-hydrogen protein atoms of 221 amino acids and 79 water molecules. PSP-I/PSP-II represents the first crystal structure of a mammalian zona pellucida-binding protein. PSP-II displays a putative carbohydrate-recognition site located around its Asn50. This region shares structural features with sugar binding sites of known lectin structures of the leguminous and galectin families. PSP-I and PSP-II are N-glycosylated at asparagine residues 50 and 98, respectively, and show site heterogeneity. Only the innermost N-acetylglucosamine of PSP-I is defined in the crystal structure. Both subunits of the PSP-I/PSP-II heterodimer are built by a single CUB domain architecture. The CUB domain displays a novel fold, which consists of a compact ellipsoidal beta-sandwich structure (42 A x 27 A x 23 A) organized into two 5-stranded beta-sheets. Each sheet contains parallel and antiparallel beta-strands. Two disulphide bridges, which are conserved in all spermadhesin molecules and many CUB domains, crosslink loop LA and strand beta 4 and loops LE and LG, respectively, at opposite edges of the same face of the domain. The four highly conserved aromatic residues and 15 out of 17 invariant hydrophobic residues, which define the CUB domain signature, display an interior location, suggesting that this hydrophobic core may be essential for maintaining the overall folding of the domain. Most of the hydrophobic core residue characteristics are conserved in the jellyroll topology of certain icosahedral virus capsid proteins, indicating that the CUB domain and the viral proteins share a minimal structural core.

Amino Acid Sequence↗
PubMed1998

Derivation of the three-dimensional architecture of bacterial ribonuclease P RNAs from comparative sequence analysis.

The secondary structure of bacterial RNase P RNA, a ribozyme responsible for the maturation of the 5' end of tRNAs, is well established on the basis of sequence comparison analysis. RNase P RNA secondary structures fall into two types, A and B, which share a common core formed by the assembly of two main folding domains, but differ in their peripheral elements.A revised alignment of 137 available sequences reveals new covariations allowing for the refinement of both types of secondary structures. Phylogenetic evidence is thus provided for the extension of stems P11, P14, P19, P10.1 and P15.1 through further canonical base-pairs or GAellipsisGA mismatches. These refinements led in turn to a new organization of the catalytic core, with coaxial stackings of helices P2 and P19 as well as P1 and P4. New inter-domain tertiary interactions involve loop L9 and helix P1 and loop L8 with helix P4. These features were incorporated into atomic-scale 3D models of RNase P RNA for representatives of each structural type, namely Escherichia coli and Bacillus subtilis. In each model, the juxtaposition of the core helices creates a cradle onto which the pre-tRNA substrate binds with most evolutionarily conserved residues converging towards the cleavage site. The inner cores of both types are stabilized similarly, albeit by different peripheral elements, emphasizing the modular and hierarchical organisation of the architecture of RNase P RNAs. Similarities are thus apparent between the type A modules, P16/P17/P6 and P13/P14, and their type B analogs, P5.1/P15.1 and P10. 1/P10.1a, respectively. Other noteworthy features of these models include compactness and good agreement with published crosslinking data.

Bacteria↗
PubMed1998

Molecular architecture of the c-subunit oligomer in the membrane domain of F-ATPases probed by tryptophan substitution mutagenesis.

Subunit c of the proton-transporting ATP synthase of Escherichia coli forms an oligomeric complex in the membrane domain that functions in transmembrane proton conduction. In order to gain some insight into the architecture of this oligomeric complex, the transmembrane region in the C-terminal membrane-spanning segment was analysed by a site-directed mutagenesis approach. Tryptophan substitution of consecutive residues in positions 61 to 72 of subunit c was used to identify residues oriented towards a helix-helix surface or an accessible phase in the oligomeric complex. Mutants were analysed in functional assays of ATP hydrolysis, ATP synthesis and ATP-dependent proton transport. Function was disrupted according to a pattern that identified inter- and intramolecular contacts in the c-subunit oligomer. Screening experiments on minimal medium support the helix-helix contacts found in the functional assays. The results add strong constraints to the potential orientation of the monomers in the oligomeric complex and are discussed against the background of different structural models that have been proposed for the c-subunit oligomer.

Adenosine Triphosphate↗
PubMed1999

Domain architecture of the bacteriophage phi29 connector protein.

Viral connectors are essential components of the DNA packaging machinery. They interact with nucleic acids and other viral components to translocate DNA inside the viral head. We have attempted to locate the different structural and functional domains of the phage Phi29 connector using a combination of approaches to generate different antigenic probes. Complexes of native connectors with either monoclonal or monospecific antibodies were studied by immunoelectron microscopy and image averaging methods. The data were merged in a model of the connector domain structure at 2-3 nm resolution. This epitope mapping provides a general outline of the folding architecture of the connector polypeptide, following a complicated threading that places the amino and carboxyl-terminals in close alignment in the narrower domain at 2-3 nm from the top of the connector. The appendages are built up by a long and highly immunogenic sequence (amino acid residues 153 to 206). The RNA binding domain forms part of the top of the narrow conical area of the connector, a flexible region that undergoes structural changes during viral morphogenesis. The DNA binding domain is located not far away, 2-3 nm below, in the outer side of the narrow conical part. The precise location of the functional domains of the connector, as well as their relative positions provide the first experimental framework for understanding the connector function.

Amino Acid Sequence↗
PubMed1999

Novel molecular architecture of the multimeric archaeal PEP-synthase homologue (MAPS) from Staphylothermus marinus.

The phosphoenolpyruvate (PEP)-synthases belong to the family of structurally and functionally related PEP-utilizing enzymes. The only archaeal member of this family characterized thus far is the Multimeric Archaeal PEP-Synthase homologue from Staphylothermus marinus (MAPS). This protein complex differs from the bacterial and eukaryotic representatives characterized to date in its homomultimeric, as opposed to dimeric or tetrameric, structure. We have probed the molecular architecture of MAPS using limited proteolytic digestion in conjunction with electron microscopic, biochemical, and biophysical techniques. The 2.2 MDa particle was found to be organized in a concentric fashion. The 93.7 kDa monomers possess a pronounced tripartite domain structure and are arranged such that the N-terminal domains form an outer shell, the intermediate domains form an inner shell, and the C-terminal domains form a core structure responsible for the assembly into a multimeric complex. The core domain was shown to be capable of assembling into the native multimer by recombinant expression in Escherichia coli. Deletion mutants as well as a synthetic peptide were investigated for their state of oligomerization using native polyacrylamide gel electrophoresis, molecular sieve chromatography, analytical ultracentrifugation, circular dichroism (CD) spectroscopy, and chemical cross-linking. Our data confirmed the existence of a short C-terminal, alpha-helical oligomerization motif that had been suggested by multiple sequence alignments and secondary structure predictions. We propose that this motif bundles the monomers into six groups of four. An additional formation of 12 dimers between globular domains from different bundles leads to the multimeric assembly. According to our model, each of the six bundles of globular domains is positioned at the corners of an imaginary octahedron, and the helical C-terminal segments are oriented towards the centre of the particle. The edges of the octahedron represent the dimeric contacts. Phylogenetic analysis suggests that the ancient predecessor of this family of enzymes contained the C-terminal oligomerization motif as a feature that was preserved in some hyperthermophiles.

Amino Acid Sequence↗
PubMed1999

Stitching together RNA tertiary architectures.

The powerful explanatory paradigm of molecular biology requiring form to co-evolve with function has again been proven successful when, over the recent two decades, a wealth of biological functions have been uncovered for RNA. Previously considered as a mere mediator of the genetic code, RNA is now acknowledged as a key player in a wide variety of cellular processes. Along with the discovery of novel biological functions of RNA molecules, a number of RNA three-dimensional structures have been solved which beautifully demonstrate the molecular adaptability which allows RNA to participate as a key player in these functions. A distinct repertoire of molecular motifs provides a basis for the assembly of complex RNA tertiary architectures.

Base Pair Mismatch↗
PubMed2001

Non-alpha-helical elements modulate polytopic membrane protein architecture.

In "all alpha-fold" transmembrane proteins, including ion channels, G-protein-coupled receptors (GPCRs), bacterial rhodopsins and photosynthetic reaction centers, relatively long alpha-helices, straight, curved or kinked, pack into compact elliptical or circular domains. Using both existing and newly developed tools to analyze transmembrane segments of all available membrane protein three-dimensional structures, including that very recently elucidated for the GPCR, rhodopsin, we report here the finding of frequent non-alpha-helical components, i.e. 3(10)-helices ("tight turns"), pi-helices ("wide turns") and intrahelical kinks (often due to residues other than proline). Often, diverse helical types and kinks concatenate over long segments and produce complex inclinations of helical axis, and/or diverse frame shifts in the "canonical", alpha-helical side-chain pattern. Marked differences in transmembrane architecture exist even between seemingly structurally related proteins, such as bacteriorhodopsin and rhodopsin. Deconvolution of these non-canonical features into their composite elements is essential for understanding the pleiotropy of polytopic protein structure and function, and must be considered in developing valid macromolecular models.

Amino Acid Sequence↗
PubMed1999

Induction of high mobility group I architectural transcription factors in proliferating vascular smooth muscle in vivo and in vitro.

Proliferation of vascular smooth muscle cells (VSMCs) is a hallmark of arteriosclerosis. Architectural transcription factors of the high mobility group (HMG)-I family have been implicated in the control of cell proliferation and gene expression. We studied the pattern of HMG-I mRNA and protein expression in proliferating VSMCs. HMG-I(Y) and HMGI-C mRNAs were barely detectable by Northern analysis in samples prepared from uninjured rat carotid arteries. In contrast, these mRNAs were induced dramatically in carotid arteries 2 and 5-6 days after balloon injury. By in situ hybridization at 6 days after injury, the induced mRNAs localized to smooth muscle cells of the developing neointima, and immunocytochemical analysis showed that HMG-I(Y) protein was expressed in the nuclei of these cells. To confirm this association between HMG-I protein induction and cell growth, we assessed HMG-I(Y) and HMGI-C mRNA expression in rat aortic smooth muscle cells (RASMCs) in primary culture. The HMG-I mRNAs were barely detectable in quiescent RASMCs but were induced markedly by serum stimulation. This induction of mRNA by serum was time dependent and peaked at 9 h. Western blot analysis confirmed that HMG-I(Y) protein induction also occurred in vitro. To our knowledge, this is the first demonstration of induction of HMG-I protein expression in proliferating RASMCs in vivo and in vitro. This demonstration suggests that the HMG-I proteins may play an important role in smooth muscle cell proliferation.

Animals↗