The in vitro assembly of crystalline gap junctions from detergent-solubilized sheep lens fiber cell pore complexes in the presence of endogenous lipid is a two-step process. "Mini"-gap junctions containing about 10 pore complexes assembled within 12 hr of dialysis. The removal of detergent appeared to be the sole factor required to reach this level of assembly. Further growth to micrometer-sized crystalline gap junctions required cleaved connexin, MgCl2, elevated dialysis temperature, and dialysis for 3-5 days. Molecular interactions between adjacent pore structures laterally in the membrane are vital for the second phase of assembly but appear to be of minor importance for the formation of mini-gap junctions. Parallels exist between structural aspects of gap junction assembly in vitro and the 2-D crystallization of membrane proteins in general.

Animals↗

PubMedDate not supplied

Direct in situ structural analysis of recombinant outer membrane porins expressed in an OmpA-deficient mutant Escherichia coli strain.

An OmpA-deficient mutant of an OmpF/OmpC-free Escherichia coli B strain was selected using phage K3. The mutant strain was characterized by SDS-gel electrophoresis, immunoblotting, and electron microscopy. All major outer membrane proteins, including OmpA, were absent. This strain was then transformed with the plasmid pMY222 encoding the K12 OmpF porin or with pBlue-script-derived plasmids, encoding the porins OmpC, PhoE, and maltoporin, respectively. Following SDS extraction of outer membrane sacculi from strains expressing individual porins, crystalline porin arrays that allowed in situ structural analysis to be performed were observed. Furthermore, the absence of endogenous major outer membrane proteins facilitated the purification of native porin-lipopolysaccharide complexes, the functionally active channels, from the sacculi of transformed strains.

Bacterial Outer Membrane Proteins↗

PubMedDate not supplied

Structural and physicochemical analysis of the contractile MM phage tail and comparison with the bacteriophage T4 tail.

The three-dimensional (3-D) structure of the bacteriophage MM extended tail has been determined from electron micrographs of negatively stained specimens and compared with 3-D models of coprocessed extended bacteriophage T4 tails. Accordingly, the phage MM extended tail exhibits an axial repeat of 3.8 nm and can be indexed according to the integer helical selection rule l = -3n + 7m (n = 6n') compared to 4.1 nm and l = -2n + 7m (n = 6n') for the T4 phage tail. Compared to the T4 tail sheath, which reveals a stacked-disk-like appearance, the MM tail exhibits a more open structure, yielding an arrow-head-like appearance. Although the phage MM extended tail sheath is more stable than the T4 tail sheath under low-ionic-strength conditions, various chemical treatments of the MM tail sheath revealed responses, notably disassembly and contraction, similar to those previously described for the T4 tail sheath. Extended tails and their structural components contained in phage lysates or prepared by chemical degradation were compared in the EM, and the mass-per-length values of extended tails and tail tubes were determined by quantitative scanning transmission electron microscopy and compared to the corresponding values computed from the respective 3-D mass density maps. Accordingly, masses of 111 and 135 kDa/nm were obtained for the MM and T4 phage tail sheaths, respectively, with the corresponding tail tubes calculated at 19.3 and 25.5 kDa/nm, respectively. Although negative staining and freeze drying/metal shadowing of the two tails revealed different extended tail sheath structures, freeze-dried/metal-shadowed specimens of their contracted tails revealed very similar 6-fold symmetric axial repeats, with the subunits arranged on a pseudo-12-fold symmetric surface lattice following the integer helical selection rule l = n + 11m. In both cases tail contraction started at the baseplate and propagated headward as a wave forming a contraction gradient with a sharp boundary.

Bacteriophage T4↗

PubMedDate not supplied

Real-time isocontouring and texture mapping meet new challenges in interactive molecular graphics applications.

A technique to extract solid isosurfaces from three-dimensional electron density data at high speed is presented. The ability to change the contouring threshold in real time renders the method a powerful tool for interactive analysis of proteins and their supramolecular assemblies, in order to compare and combine structural information gathered by different data acquisition methods. Chemical properties can effectively be mapped onto these isosurfaces by the use of texture mapping. The implications of these methods in combination with other visualization techniques are discussed, and a framework for their integration into a general-purpose molecular graphics toolkit is proposed.

Carbohydrate Conformation↗

PubMedDate not supplied

Proposal for a new distributed database of macromolecular and subcellular structures from different areas of microscopy.

In this work we address the problem of information access that arises in the field of three-dimensional structure determination, by means of image processing, from data obtained by various types of microscopy. A prototype of a distributed database containing three-dimensional structural information is presented. In this database the volume information is linked, if possible, to other sources of catalogued information such as sequence data, atomic coordinates, and bibliographies. The solution we propose is sufficiently general to be applicable to data in other fields of biomedical science.

Computer Communication Networks↗

PubMedDate not supplied

Structural studies on the 2.25-MDa homomultimeric phosphoenolpyruvate synthase from Staphylothermus marinus.

The phosphoenolpyruvate synthase of the hyperthermophilic archaeon Staphylothermus marinus forms an unusually large homomultimeric complex of 93 kDa subunits. Electron image analysis of negatively stained and low-dose unstained preparations showed that the complex has a single, stable characteristic view and a well-preserved core with threefold rotational symmetry. The periphery of the assembly is composed of a nebulous, possibly flexible, component. Mass measurements by scanning transmission electron microscopy yielded a molecular weight of 2250 +/- 230 kDa, confirming the well-defined nature of the structure and indicating that it is composed of 24 +/- 2.5 subunits. The stability and symmetry of the characteristic projection views suggest a polyhedral three-dimensional architecture. The novel quaternary arrangement of this enzyme might be a consequence of its adaptation to an extreme environment.

Archaea↗

PubMedDate not supplied

Surface and subsurface morphology of bovine humeral articular cartilage as assessed by atomic force and transmission electron microscopy.

Maintenance of superficial structural integrity is essential for the load-bearing function of articular cartilage. In this study, we used atomic force microscopy (AFM) to image the 3-D surface and subsurface morphology of fresh bovine humeral head articular cartilage maintained in physiological solution. Complementary ultrastructural data were obtained by transmission electron microscopy (TEM) of cryo-processed samples. The surface irregularities observed in previous scanning electron microscopic studies were not apparent with AFM. The most superficial layer, typically 200-500 nm thick, consisted of acellular and nonfibrous tissue. Occasionally, it exhibited local discontinuities through which the underlying network of collagen fibrils, oriented parallel to the surface and displaying the characteristic periodic banding, could been seen. Local variations in force-curve measurements indicate the existence of differences in micromechanical properties along the articular surface. AFM thus furnishes a new method for characterizing the surface structure and properties of freshly excised articular cartilage in physiologically relevant conditions. It confirms the existence of an amorphous, nonfibrous articular surface which may be vital for its normal lubrication and wearing properties in vivo.

Animals↗

PubMedDate not supplied

Visualizing 3D data obtained from microscopy on the Internet.

The Internet is a powerful communication medium increasingly exploited by business and science alike, especially in structural biology and bioinformatics. The traditional presentation of static two-dimensional images of real-world objects on the limited medium of paper can now be shown interactively in three dimensions. Many facets of this new capability have already been developed, particularly in the form of VRML (virtual reality modeling language), but there is a need to extend this capability for visualizing scientific data. Here we introduce a real-time isosurfacing node for VRML, based on the marching cube approach, allowing interactive isosurfacing. A second node does three-dimensional (3D) texture-based volume-rendering for a variety of representations. The use of computers in the microscopic and structural biosciences is extensive, and many scientific file formats exist. To overcome the problem of accessing such data from VRML and other tools, we implemented extensions to SGI's IFL (image format library). IFL is a file format abstraction layer defining communication between a program and a data file. These technologies are developed in support of the BioImage project, aiming to establish a database prototype for multidimensional microscopic data with the ability to view the data within a 3D interactive environment.

Animals↗

PubMedDate not supplied

Soluble tumor necrosis factor receptors in febrile neutropenic cancer patients.

Explore the source record for details and available documents.

Antineoplastic Agents↗

PubMedDate not supplied

Kinetics and correlation with body temperature of circulating interleukin-6, interleukin-8, tumor necrosis factor alpha and interleukin-1 beta in patients with fever and neutropenia.

Interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF) are important mediators of fever and inflammation, and are involved in the pathogenesis of sepsis. There is only limited data on serum concentrations of these proinflammatory cytokines in patients with fever and neutropenia, and their interrelationship and correlation with body temperature and clinical disease early in the febrile response during neutropenia have not been studied. Immunoreactive TNF, IL-1 beta, IL-6, and IL-8 in serum samples serially obtained from 14 adult patients with neutropenia and fever considered or documented to be due to infection were measured. IL-6 and Il-8 were consistently elevated in all patients, and correlated well with each other and with body temperature. Median peak concentration of IL-6 and IL-8 were 400 pg/ml (range: 100 to 41,000 pg/ml), and 1,025 pg/ml (range: 600 to 26,000 pg/ml), respectively, and levels of both cytokines rapidly declined in patients responding to antimicrobial therapy. Despite frequent sampling before and after the temperature peaks TNF and IL-1 beta, conversely, were less frequently detectable, with median peak values of < 10 pg/ml (range: < 10 to 150 pg/ml) for TNF, and 17 pg/ml (range: < 10 to 36 pg/ml) for IL-1 beta, respectively. The role of neutro- and monocytopenia with depletion of important cytokine producing and target cells in this particular cytokine response pattern needs to be further studied.

Acute Disease↗

PubMedDate not supplied

Subclavian artery aneurysm caused by cervical rib: case report and review.

An aneurysm in the subclavian artery is relatively uncommon and a cervical rib as the cause is rare. Such a case is presented and the pertinent literature is reviewed. The mechanism of aneurysm formation in a normal vessel wall is discussed.

Aged↗