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In vivo control of endosomal architecture by class II-associated invariant chain and cathepsin S.

The invariant chain (Ii) is a chaperone that regulates assembly and transport of class II MHC molecules. In the absence of the lysosomal protease cathepsin S (CatS), degradation of Ii is impaired and an Ii remnant that extends from the N terminus to about residue 110 accumulates in class II MHC-positive endosomal compartments, which are enlarged in size and lack multivesicular morphology. In primary B cells examined in vitro and in lymph nodes examined by immuno-electron microscopy, CatS controls architecture of class II-positive endosomal compartments. In a compound mutant mouse that lacks both CatS and Ii, the normal size of endosomes in class II-positive cells is restored, although internal endosomal membranes are absent. Proper degradation of Ii is thus essential for normal endosomal morphology in antigen-presenting cells in vivo.

Animals↗

Interchange and intra-nuclear architecture.

For chromatin lesions to interact to form exchanges of any sort, it is obvious that contact between them must be made. However, the probability of such interaction is conditioned by other factors like time, initial separation, metabolic activity, and, in the case of chemically induced lesions, scheduled DNA synthesis. The irradiated nucleus was, for a long time, regarded as a "bag of broken chromosomes" with the severed ends free to move around and find partners with which to form illegitimate reunions. Many of these would be seen at following metaphase as intra- and interchanges. Evidence is rapidly accumulating which indicates that this picture of the nucleus is false. We know now that chromosomes occupy highly localised domains with limited movement, and that there is no massive intermingling; that much of the chromatin is compacted and splinted with proteins and so precluded from exchange-type contact; that most of the chromatin is looped and "fixed" into an intra-nuclear protein scaffold or skeleton; that some chromatin is spun-out and associated with the nuclear envelope in the vicinity of the pore-complexes. Thus it would appear that movement, in the sense envisaged by early workers, is curtailed, and that only a proportion (probably a small proportion) of the chromatin is actually "at-risk" with respect to interchange formation. Where then does interchange take place? Are the "sites" pre-existent, or can proximity requirements be realised after radiation exposure? In what ways will the intra-nuclear architecture influence exchange? These are some of the questions which are considered in this paper.

Animals↗

HMG-D is an architecture-specific protein that preferentially binds to DNA containing the dinucleotide TG.

The high mobility group (HMG) protein HMG-D from Drosophila melanogaster is a highly abundant chromosomal protein that is closely related to the vertebrate HMG domain proteins HMG1 and HMG2. In general, chromosomal HMG domain proteins lack sequence specificity. However, using both NMR spectroscopy and standard biochemical techniques we show that binding of HMG-D to a single DNA site is sequence selective. The preferred duplex DNA binding site comprises at least 5 bp and contains the deformable dinucleotide TG embedded in A/T-rich sequences. The TG motif constitutes a common core element in the binding sites of the well-characterized sequence-specific HMG domain proteins. We show that a conserved aromatic residue in helix 1 of the HMG domain may be involved in recognition of this core sequence. In common with other HMG domain proteins HMG-D binds preferentially to DNA sites that are stably bent and underwound, therefore HMG-D can be considered an architecture-specific protein. Finally, we show that HMG-D bends DNA and may confer a superhelical DNA conformation at a natural DNA binding site in the Drosophila fushi tarazu scaffold-associated region.

Animals↗

Control of in vivo microvessel ingrowth by modulation of biomaterial local architecture and chemistry.

We developed a method for controlling local architecture and chemistry simultaneously in biomaterial implants to control microvessel ingrowth in vivo. Porous polypropylene disks (5 mm in diameter and 40 microm thick) were plasma-coated with a fluoropolymer and then laser-drilled with 50-microm-diameter holes through their thickness. We then oxidized the disks to create hydroxyl functionality on the exposed polypropylene (inside the holes). Acrylamide was grafted to the hydroxyl groups through polymerization in the presence of activating ceric ions. Staining with toluidine blue O demonstrated that grafting occurred only inside the holes. We used the Hoffman degradation reaction to convert the amide groups of acrylamide to amine groups, and then we used ethylene glycol diglycidyl ether to attach biomolecules of interest inside the holes: secreted protein acidic and rich in cysteine (SPARC) peptide Lys-Gly-His-Lys (KGHK; angiogenic), thrombospondin-2 (TSP; antiangiogenic), or albumin (rat; neutral). In vivo testing in a rat subcutaneous dorsum model for a 3-week interval demonstrated a greater vessel surface area (p = 0.032) and a greater number of vessels (p = 0.043) in tissue local to the holes with KGHK-immobilized disks than with TSP-immobilized disks. However, differences between KGHK-immobilized and albumin-immobilized disks were less significant (p = 0.120 and p = 0.289 for the vessel surface area and number of vessels, respectively). The developed methods have potential applications in biomaterial design applications for which selective neovascularization is desired.

Animals↗

Design and fabrication of standardized hydroxyapatite scaffolds with a defined macro-architecture by rapid prototyping for bone-tissue-engineering research.

This investigation describes the production and characterization of calcium phosphate scaffolds with defined and reproducible porous macro-architectures and their preliminary in vitro and in vivo bone-tissue-engineered response. Fugitive wax molds were designed and produced using a rapid prototyping technique. An aqueous hydroxyapatite slurry was cast in these molds. After sintering at 1250 degrees C and then cleaning, dimensional and material characterizations of the scaffolds were performed. The resulting scaffolds represented the design, and their dimensions were remarkably consistent. A texture inherent to the layer-by-layer production of the mold was impressed onto the vertical surfaces of the scaffolds. The surface roughness (R(a)) of the textured surfaces was significantly greater than that of the nontextured surfaces. Material analyses revealed a beta-TCP phase in addition to hydroxyapatite for the molded ceramics. Non-molded control ceramics exhibited only hydroxyapatite. Thirty scaffolds were seeded with culture-expanded goat bone-marrow stromal cells (BMSCs) and implanted subcutaneously in nude mice for 4 or 6 weeks. Histology revealed mineralized bone formation in all the scaffolds for both implantation periods. After 4 weeks, bone was present primarily as a layer on scaffold surfaces. After 6 weeks, the surface bone formation was accompanied by bone budding from the surface and occasional bridging of pores. This budding and bridging bone formation almost always was associated with textured scaffold surfaces. However, the area percentage of bone in pores was similar for the 4- and 6-week implantation periods.

Animals↗

Bioactive glass as precursor of designed-architecture scaffolds for tissue engineering.

In this work, the conditions to obtain concentrated and fluid suspensions from a bioactive glass (55-SiO(2); 41-CaO; 4-P(2)O(5); mol %) were investigated. The influence of the heat treatment of the glass on the specific surface area, solubility, bioactivity, and finally on their dispersion characteristics was studied. Zeta potential and viscosity measurements were carried out, and based on the obtained results, the best dispersant was selected. The optimum concentration of dispersant, maximum content of solid and time of mixing were also investigated. Slurries containing 50 vol % could be obtained calcining the glass at 1100 degrees C and using Darvan 811 (sodium polyacrylate) as dispersant. Scaffolds with designed architecture were prepared from these suspensions combining the gelcasting method and the stereolithography technique. A polymeric negative (replica of the desired structure) was previously obtained by stereolithography. The slurry was cast into the molds and then polymerized (gelcasting method). The negative was eliminated by heat treatment. After sintering at 1300 degrees C, scaffolds with interconnected porosity and three-dimensional channels of 400-470 microm and macropores of 1.4 microm were obtained.

Ceramics↗

Polymer scaffolds with interconnected spherical pores and controlled architecture for tissue engineering: fabrication, mechanical properties, and finite element modeling.

A method is proposed in which the geometric properties of 3D scaffolds with application in tissue engineering can be tailored: porosity, pore size, and interconnection throat size. The architecture of the fabricated scaffolds is analyzed by scanning electron microscopy. The mechanical properties of these structures are discussed on the basis of compression stress-strain measurements. Moreover, the mechanical properties of the scaffolds are estimated by means of finite element modeling (FEM) in which the compression stress-strain test is simulated on an ideal structure based on the crystalline face centered cubic system. The elastic properties of the constructs are explained on the basis of the FEM model that supports the main mechanical conclusion of the experimental results: the compressive modulus in the first linear region does not depend on the geometric characteristics of the pore (pore size, interconnection throat size) but only on the total porosity of the scaffold.

Biocompatible Materials↗

The direct examination of three-dimensional bone architecture in vitro by computed tomography.

We describe a new method for the direct examination of three-dimensional bone structure in vitro based on high-resolution computed tomography (CT). Unlike clinical CT, a three-dimensional reconstruction array is created directly, rather than a series of two-dimensional slices. All structural indices commonly determined from two-dimensional histologic sections can be obtained nondestructively from a large number of slices in each of three orthogonal directions. This permits a comprehensive description of structural variation within a specimen and greatly facilitates the study of structural anisotropy. A measure of three-dimensional connectivity (Euler number/tissue volume) has been determined for the first time in human cancellous bone and shown to correlate with several two-dimensional histomorphometric indices. The method has the potential for overcoming many of the limitations of current approaches to the study of bone architecture at the microscopic level.

Bone and Bones↗

Modifications of nuclear architecture and chromatin organization in ataxia telangiectasia cells are coupled to changes of gene transcription.

Ataxia telangiectasia (AT) is a rare genetic disorder caused by mutations of ATM gene. ATM kinase is a "master controller" of DNA-damage response and signal transducer of external stimuli. The complex role of ATM may explain the pleiotropic phenotype characteristic of AT syndrome, only partially. In our hypothesis, the multi-faceted phenotype of AT patients might depend on specific chromatin reorganization, which then reflects on the cellular transcription. We analyzed three lymphoblastoid cell-lines isolated from AT patients and one healthy control. The three-dimensional reconstruction disclosed marked changes of nuclear morphology and architecture in AT cells. When chromatin condensation was analyzed by differential scanning calorimetry, a remodeling was observed at the level of fiber folding and nucleosome conformation. Despite the structural differences, chromatin did not exhibit modifications of the average acetylation status in comparison to the control. Moreover, AT cells presented significant alterations in the transcription of genes involved in cell-cycle regulation and stress response. In AT3RM cells, the average chromatin decondensation went with the upregulation of c-fos, c-jun, and c-myc and downregulation of metallothioneins, p21 and p53. AT9RM and AT44RM cells were instead characterized by an increased chromatin condensation and presented a different transcription unbalance. Whereas in AT44RM all the considered genes were downregulated, in AT3RM the three oncogenes and metallothioneins were upregulated, but p53 and p21 were downregulated.

Acetylation↗

A novel parallel algorithm for large-scale Fock matrix construction with small locally distributed memory architectures: RT parallel algorithm.

We developed a novel parallel algorithm for large-scale Fock matrix calculation with small locally distributed memory architectures, and named it the "RT parallel algorithm." The RT parallel algorithm actively involves the concept of integral screening, which is indispensable for reduction of computing times with large-scale biological molecules. The primary characteristic of this algorithm is parallel efficiency, which is achieved by well-balanced reduction of both communicating and computing volume. Only the density matrix data necessary for Fock matrix calculations are communicated, and the data once communicated are reutilized for calculations as many times as possible. The RT parallel algorithm is a scalable method because required memory volume does not depend on the number of basis functions. This algorithm automatically includes a partial summing technique that is indispensable for maintaining computing accuracy, and can also include some conventional methods to reduce calculation times. In our analysis, the RT parallel algorithm had better performance than other methods for massively parallel processors. The RT parallel algorithm is most suitable for massively parallel and distributed Fock matrix calculations for large-scale biological molecules with more than thousands of basis functions.

Algorithms↗

An architectural perspective of cell-cycle control at the G1/S phase cell-cycle transition.

A prominent role for the execution of cell cycle and growth regulatory mechanisms within the three-dimensional context of nuclear architecture is becoming increasingly evident. Signaling pathways and regulatory networks that govern activation and suppression of genes controlling proliferation are functionally integrated for the organization and assembly of transcriptional machinery in nuclear microenvironments. The transcriptional activation of histone genes at the G1/S phase transition (S-point) is temporarily, functionally, and spatially distinct from transcriptional mechanisms at the restriction point (R-point). The spatial distinction in R-point versus S-point control is the localization of clustered histone gene loci at cajal bodies, which is modulated during the cell cycle. Histone nuclear factor P (HiNF-P), the principal factor mediating H4 histone gene transcription, is the final link in the signaling cascade that is initiated with growth factor dependent induction of cyclin E/CDK2 kinase activity at the R-point and culminates in the NPAT-mediated activation of histone H4 genes through HiNF-P at the G1/S phase cell-cycle transition.

Cell Cycle↗

Computer reconstruction of nucleolar architecture by interactive three-dimensional color display.

The first complete three dimensional ultrastructural reconstruction of pancreatic cell nucleoli, was done using EM and computer 3D-assisted reconstruction of serial sections with interactive 3D back-to-front and color display methods based on voxel representation. The purpose of the study was to depict the architecture of the nucleolar components. We obtained information about the location of the nucleolus within the nuclear volume and about the shape and polarity of the 3 main nucleolar territories.

Animals↗

Leishmaniosis--a report about the microvascular and cellular architecture of the infected spleen in Canis familiaris.

Leishmaniosis is an anthropozoonosis caused by an intracellular protozoan parasite that causes a wide spectrum of diseases in humans and dogs worldwide. In the Mediterranean basin, Portugal, Central and South America, and in the Middle East, visceral leishmaniosis is caused by Leishmania infantum. In these areas, dogs are believed to be the natural reservoirs of this parasite. In the case of visceral leishmaniosis, the spleen is one of the several hematopoietic and immunocompetent organs involved. Since this viscera is a blood filter, the authors investigated the expression of the morphological and microvascular environment and modifications of the spleen cell population related to immunological responses to this parasitic condition. The tools used to perform this study were scanning electronic microscopy of intact tissue and corrosion casts, transmission electronic microscopy, histology and immunohistochemistry. The results reveal three important modifications concerning the spleen's microvascular architecture when compared with its normal pattern, independently of the serological titer obtained with indirect immunofluorescence. (1) A marked scarcity of the sinusoidal system sheet that surrounds the central artery/arteriole of the white pulp; (2) A huge development of pulp venules and veins; (3) The presence of a surprising development of reticular fibers. The authors postulate that independent of the virulence of the parasite involved and the type of immunity prevalent in a particular host, the spleen develops blood dynamic conditions that permit reduction in the speed of blood flow so that cells involved in immunological processes can proliferate and differentiate, and also contributes to trapping lymphocytes within the area through the differentiation of characteristics that resemble those of HEV endothelial cells.

Animals↗

Vascular architecture and intestinal hematopoietic nests of two cyclostomes, Eptatretus burgeri and ammoncoetes of Entosphenus reissneri: a comparative morphological study.

Vascular architecture and the structure of the intestinal hematopoietic centers of two cyclostomes, the hagfish Eptatretus burgeri and the ammocoetes larva of Entosphenus reissneri, are compared. Blood cells of the hagfish are generated in hematopoietic nests that develop around intestinal veins established primarily for transport of absorbed nutrients. In ammocoetes, on the other hand, blood cells are generated in hematopoietic nests of the typhlosole, closely associated with venous sinusoids developing around the longitudinally oriented mesenteric artery of the typhlosole. A collateral vein of the mesenteric artery is completed in the typhlosole after metamorphosis. Since the spleen of higher vertebrates develops in relation to establishment of the collateral vein of the largest foregut artery, the intestinal hematopoietic nests of ammocoetes may be regarded as a model of the primitive form of the spleen of higher vertebrates. Hematopoiesis in the hagfish intestine is not related to establishment of a collateral vein; hence "primitive spleen" or "intestinal spleen" may be improper terms in reference to the intestinal hematopoietic tissue of the hagfish. Morphological characteristics of the hematopoietic nests of the two cyclostomes are essentially the same. Blood cells of these nests are generated in the intervenous tissue, supported by interstitial connective tissue cells and reticulin fibers. Granulated cells are the most common type in the primitive hematopoietic nests. No definitive erythrothrombocytopoiesis has been identified. Lymphocytes have not been observed in the hagfish; however, small lymphocytes have been observed in the vascular lumen of sinusoids around the hematopoietic nests of ammocoetes. These lymphocytes probably originate outside of the typhlosole.

Animals↗

Muscle-fiber architecture, innervation, and histochemistry in the diaphragm of the cat.

Previous anatomical descriptions of the diaphragm have contained several contradictory findings. To validate and extend the previous work, diaphragmatic architecture, histochemistry, and end-plate distribution were examined by use of a combination of anatomical methods, including fiber microdissections, cholinesterase staining, and enzyme histochemistry. Microdissections showed that muscle-fiber fascicles throughout the diaphragm contain both long fibers that run from origin to insertion and shorter fibers with intrafascicular terminations. Fibers with intrafascicular terminations were particularly common in the costal diaphragm, where they accounted for the majority of sampled fibers. The heterogeneity of fiber length was reflected in the pattern of end-plate banding. Cholinesterase studies showed that fiber fascicles in cat and kitten diaphragms were crossed by two to four end-plate bands distributed in discontinuous arrays across the width of the muscle. A similar pattern of multiple banding was also demonstrated in the adult and neonatal dog. However, rat and rabbit diaphragms had only a single, continuous end-plate band. Histochemical studies of fiber types in different parts of the feline diaphragm showed that costal, crural, and sternal subregions had similar overall proportions of fiber types. However, type SO (slow oxidative) fibers were distributed more densely on the thoracic than the abdominal surface of costal and crural, but not sternal subregions. Type SO fibers were also concentrated in fiber fascicles bordering the esophageal hiatus.

Animals↗

Three-dimensional architecture of identified cerebral neurosecretory cells in an insect.

The organization of identified neurosecretory cell groups in the larval brain of the tobacco hornworm, Manduca sexta, was investigated immunocytologically. Computer-assisted three-dimensional reconstruction was used to examine the architecture of the neurosecretory cell groups. The group III lateral neurosecretory cells (L-NSC-III) which produce the prothoracicotropic hormone are located dorsolaterally in the protocerebrum and extend axons medially that decussate to the contralateral lobe prior to exiting the brain through the nervi corporis cardiaci I + II. The group IIa2 medial neurosecretory cells (M-NSC IIa2) are located anteriorly in the medial dorsal protocerebrum. The axons of these cells also exit the brain via the contralateral nervi corporis cardiaci I + II. However, their axons traverse a different pathway through the brain from that of the L-NSC III axons. Each of the cell groups possesses elaborate dendrites with terminal varicosities. The dendrites can be classified into specific fields based upon their location and projection pattern within the brain. The dendrites for these two neurosecretory cell groups overlap in specific regions of the protocerebral neuropil. After the axons of these neurosecretory cells exit the brain through the retrocerebral nerve, they innervate the corpus allatum where they arborize to form neurohemal terminals in strikingly different patterns. The L-NSC III penetrate throughout the glandular structure and the M-NSC IIa2 terminals are restricted to the external sheath. A third group of cerebral neurosecretory cells, the ventromedial neurons (VM) which stain with the monoclonal antibody to prothoracicotropic hormone in Manduca, are located anteriorly in the medial region of the brain. The axons of these cells do not exit the brain to the retrocerebral complex, but rather pass through the circumesophageal connectives and ventral nerve cord. These neurons appear to be the same VM neurons that produce eclosion hormone. One dendritic field of the L-NSC III terminates in close apposition to the VM neurons. The distinct morphologies of these neurosecretory cell groups in relation to other cell groups and the distribution of neuropeptides within the neurons suggest that insect neurosecretory cells, like their vertebrate counterparts, may have multiple regulatory roles.

Animals↗

Bacteriophage Ø29 protein p6: an architectural protein involved in genome organization, replication and control of transcription.

Protein p6 of B. subtilis bacteriophage Ø29 binds to DNA forming a nucleoprotein complex in which the DNA wraps a protein core forming a right-handed superhelix, therefore restraining positive supercoiling and compacting the DNA. The protein does not specifically recognize a nucleotide sequence but rather a structural feature and it binds as a dimer through the minor groove. Protein p6 is in a monomer-dimer equilibrium that shifts to higher-order structures at a concentration of about 1 mM. These structures are probably present in vivo as the intracellular concentration of p6 is estimated to be in this range, and in fact the effective concentration should be still higher due to the macromolecular crowding. The p6 oligomers show an elongated shape compatible with a helical structure reminiscent of the superhelical DNA of the nucleoprotein complex, therefore it was proposed that protein p6 forms a scaffold on which the DNA folds. Since protein p6 is very abundant in infected cells, enough to bind the entire viral progeny, it was proposed to have an architectural role organizing and compacting the viral genome. It has been demonstrated that protein p6 binds in vivo to most, if not all, the Ø29 genome, although with different affinity, the highest one corresponding to the genome ends. Binding to plasmidic DNA was much lower, although it increased dramatically when the negative superhelicity was decreased. Hence, protein p6 binding specificity for Ø29 DNA is based on supercoiling, providing that the Ø29 genome, although topologically constrained, has a negative superhelicity lower than that of plasmid DNA. The formation of the nucleoprotein complex has functional implications in DNA replication and the control of transcription. It activates the initiation of replication that occurs at the genome ends for which the binding affinity is highest. It represses early transcription from promoter C2, and, together with protein p4, it represses transcription from promoters A2b and A2c and activates late transcription from promoter A3; therefore, protein p6 is involved in the early to late transcription switch.

Bacillus Phages↗

Optimal post-contrast timing of breast MR image acquisition for architectural feature analysis.

PURPOSE: To define a post-contrast imaging time span during which diagnostic accuracy of breast magnetic resonance (MR) architectural feature analysis is maintained. MATERIALS AND METHODS: Seventy-five patients with mammographically-visible or palpable findings underwent MR examination. Three sequential post-contrast, fat-saturated, three-dimensional gradient-echo imaging runs were acquired spanning 0-90, 90-180, and 180-270 seconds after contrast injection. Five readers independently predicted the malignant potential of the MR abnormalities. RESULTS: Receiver-operator characteristics (ROC) curves were our primary measure of diagnostic accuracy. The accuracy of four readers was unchanged over the three post-contrast runs. One reader was slightly more accurate using the second and third runs than using the first. CONCLUSION: For most readers, a single post-contrast run performed at any point during the first four minutes and 30 seconds following injection should yield an equivalent diagnostic accuracy. If any time period is less optimal, it is that of our first run, performed between 0-90 seconds after contrast injection.

Adult↗