Biomedical subjects
Publications and source records attributed to Y Fujiyoshi.
We encountered a 65-year-old man with tracheobronchopathia osteoplastica with "rock garden" findings on fiberoptic bronchoscopy. Many nodular elevated lesions were seen on all sides of the trachea and main bronchi except the membranous portion. The diagnosis was confirmed by examination of a biopsy specimen.
We report the structures of flagellar filaments reconstituted from various flagellins with small terminal truncations. Flagellins from Salmonella typhimurium strains SJW1103 (wild type), SJW1660, and SJW1655 were used, which form a left-handed supercoil, the L- and R-type straight forms, respectively. Structure analyses were done by electron cryomicroscopy and helical image reconstruction with a help of x-ray fiber diffraction for determining precise helical symmetries. Truncation of either terminal region, irrespective of the original flagellin species, results in a straight filament having a helical symmetry distinct either from the L- or R-type. This filament structure is named Lt-type. Although the local subunit packing is similar in all three types, a close comparison shows that the Lt-type packing is almost identical to the R-type but distinct from the L-type, which demonstrates the strong two-state preference of the subunit interactions. The structure clearly suggests that both termini are located in the inner tube of the concentric double-tubular structure of the filament core, and their proper interaction is responsible for the correct folding of fairly large terminal regions that form the inner tube. The double tubular structure appears to be essential for the polymorphic ability of flagellar filaments, which is required for the swimming-tumbling of bacterial taxis.
A variant form of the Kdp-ATPase of Escherichia coli was overproduced to a level approaching 37% of the protein in the inner membrane of this organism. Membranes from overproducing cells were prepared with an inside-out orientation. Incubation of the membranes on ice for 1-2 weeks in the presence of sodium vanadate resulted in the formation of two-dimensional crystals of the Kdp-ATPase. The calculated projection map of the p1 crystal form showed three prominent density peaks at a resolution of 22 A. This technique is a useful and simple method to obtain low-resolution structures of membrane proteins.
The cell body size and coexpression of carbonic anhydrase (CA), calretinin (CR) and calcitonin gene-related peptide (CGRP) of primary neurons with calbindin-D28k (CB) was examined in the trigeminal ganglion (TG) of the rat. CB-immunoreactive (-ir) cells were mostly large and preferentially distributed in the maxillary and mandibular divisions of the TG. 48% of CB-ir TG cells exhibited enzyme CA activity. 10% of CB-ir TG cells contained CR-ir. Most TG cells coexpressing CB- and CR-irs were localized to the maxillary and mandibular divisions and exhibited CA activity. 6.5% of CB-ir TG cells coexisted with CGRP-ir. 46% of TG cells coexpressing CB and CGRP exhibited CA activity. The innervation of the molar tooth pulp by CB-ir TG primary neurons was also examined. CB-ir thick and smooth nerve fibers projected from the root pulp to the pulp horn and the roof of the pulp chamber, where they became thinner and rarely entered the subodontoblastic layer. However, they could not be traced to the odontoblastic layer, predentin or dentine. The distribution pattern of CB-ir pulpal fibers was different from that of CR-ir ones. The trigeminal neurons cells retrogradely labeled with fast blue (FB) from the maxillary molar tooth pulp contained CB- and CR-irs. 23% and 1% of the labeled cells were immunoreactive for CB and CR, respectively. The coexpression of CB- and CR-immunoreactivities (-irs) in FB-labeled cells was negligible. An immunoelectron microscopic method revealed that 21% of pulpal nerve fibers were immunoreactive for CB, and that all CB-ir nerve fibers in the root pulp were myelinated. The present study indicated that the tooth pulp primary neurons contained CB-ir but did not coexpress CB- and CR-irs and that these neurons projected their myelinated axons to the pulp.
The supercoiled forms of the flagellar filaments are thought to be constructed from a mixture of two distinct subunit conformations arranged in a regular manner. We analyzed the structure of one of the two straight flagellar filaments, each of which is built up with all its subunits in one of the two conformations. The filament we studied was isolated from the strain SJW1655 of Salmonella typhimurium and had a right-handed helical symmetry. With recent advancements in electron cryomicroscopy, such as a liquid helium temperature stage for frozen hydrated specimens and a stable field emission source, and also by averaging high resolution data with a proper correction of the contrast transfer function, the density distribution map of this straight flagellar filament was generated in far more detail than before by including data up to 9 A resolution. The structure shows a densely packed core region from about 15 to 55 A in radius, where a pair of concentric tubular features of high density is present without well-defined subunit boundaries, and an outer part from 55 to 115 A, where the subunits are mostly well separated from each other. The outer tube in the core region, from 35 to 55 A in radius, contains many rod-like features with near-axial orientation and closest lateral distances of around 10 A, which are most likely to represent the alpha-helical bundles that were predicted in our previous report. In the inner tube, from 15 to 30 A in radius, the rod-like features are less clear. Between the inner and outer tubes are the short spoke-like densities, which are radially tilted and are connecting the two tubes. The outer part, from 55 to 115 A, contains an axially elongated column density and a slewed projection with a narrow neck region. When compared with the other straight filament having left-handed helical symmetry, this outer part does not show any significant changes in orientation, suggesting that the switch in the subunit conformation and packing involved in the polymorphic transitions is quite subtle and only occurs within the core region. Reassignment of each structural domain to the amino acid sequence is suggested, based on the volume of each domain, which was determined rather precisely by a proper correction of the contrast transfer function for both amplitudes and phases.
Two-dimensional crystals of soluble proteins were formed at the interface between an aqueous solution of proteins and a thin organic liquid (dehydroabietylamine). Proteins were adsorbed to the interface from the aqueous side and formed a two-dimensional crystal under suitable conditions. This method offers the advantage of great surface mobility and ideal homogeneity. Furthermore, the positive charge attracted negatively charged proteins well to the interface and no denaturation of the proteins was observed. With this technique, two-dimensional crystals of ferritin, catalase, chaperonin and 50S ribosome were prepared and their structural features were determined.
The structure of the light-harvesting chlorophyll a/b-protein complex, an integral membrane protein, has been determined at 3.4 A resolution by electron crystallography of two-dimensional crystals. Two of the three membrane-spanning alpha-helices are held together by ion pairs formed by charged residues that also serve as chlorophyll ligands. In the centre of the complex, chlorophyll a is in close contact with chlorophyll b for rapid energy transfer, and with two carotenoids that prevent the formation of toxic singlet oxygen.
A rapidly frozen vitrified aqueous suspension of influenza A virus was observed by high resolution electron cryomicroscopy. The influenza particles were grouped into small (diameter < 150 nm) spherical particles with well organized interiors, large spherical ones with less internal organization, and filamentous ones. Envelopes of most of the large virus particles were phospholipid bilayers, and the chromatography fraction containing these large particles was largely devoid of viral activity. The envelopes of most of the filamentous and small spherical virus particles, on the other hand, gave a strange contrast which could be ascribed to a combination of a thin outer lipid monolayer and a 7.2 nm thick protein-containing inner layer. These latter particles represented most of the viral activity in the preparation. Densitometric traces of the near in-focus images confirmed these structural differences. Some viral envelope structures apparently intermediate between these two distinct types of membrane were also detected. A structural model of intact biologically active influenza virus particles was formulated from these results, together with computer simulations.
A series of 204 amino acid proteins intended to form TIM (triose phosphate isomerase) barrel structures were designed de novo. Each protein was synthesized by expression of the synthetic gene as a fusion protein with a portion of human growth hormone in an Escherichia coli host. After BrCN treatment, the protein was purified to homogeneity. The refolded proteins are globular and exist as monomers. One of the designed proteins is stable toward guanidine hydrochloride (GuHCl) denaturation, with a midpoint of 2.6 M determined from CD and tryptophan fluorescence measurements. The GuHCl denaturation is well described by a 2-state model. The NMR spectra, the thermal denaturation curves, and the 1-anilino-8-naphthalene sulfonic acid binding imply that the stability of the protein arises mainly from hydrophobic interactions, which are probably of a nonspecific nature. The protein has a similar shape to that of rabbit triosephosphate isomerase, as determined by electron microscopy.
A new method to measure the bilayer thickness of liposomes is described. Frozen hydrated liposomes composed of either phosphatidylcholine (PC) or PC containing 30 mol% cholesterol were observed in vitreous ice with a cryo-electron microscope, which enables high-resolution observation of frozen-hydrated unstained biological specimens. The examined PCs were di-(saturated acyl) PCs (di-C12:0 (DL), diC14:0 (DM), di-C16:0 (DP)), unsaturated acyl PCs (diC18:1 (DO), C16:0-C18:1 (PO)) and PC from a natural source (egg-yolk (EY) PC). Every liposome image displayed a pair of concentric circles indicating a different bilayer structure due to the acyl-chain species. The observation of the liposomes showed the following results; (1) liposomes including unsaturated acyl PC (DOPC, POPC and EYPC) displayed a smooth and regular image of the bilayer and sharp distributions of the bilayer thickness, while the images of those composed of di-(saturated acyl) PC (DLPC, DMPC and DPPC) showed irregular shapes and wide distributions of the bilayer thickness, (2) addition of 30 mol% cholesterol to the di-(saturated acyl) PCs dispelled the irregularity in the shape of the membrane and the distribution of the bilayer thickness, and (3) the mean bilayer thickness of the liposome composed of di-(saturated acyl) PCs increased corresponding to the carbon numbers of the acyl chains. The reliability of the observations and measurements was confirmed by coupling of the cryo-electron microscopy with image simulation using the MULTI SLICE computer program and the error estimation of +/- 0.63 A using images of crystalline chlorinated copper phthalocyanine.
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An electron microscopical study was made of human immunodeficiency virus type 1 (HIV-1) in the unstained, frozen, hydrated state at liquid helium temperature. The findings were compared with those obtained from negative staining and ultrathin sectioning. Electron cryo-microscopy confirms and extended the findings obtained by conventional methods. In particular, the virus is shown to be globular, the lipid membrane is clearly resolved as a bi-layer and the bi-layer was demonstrated to be non-uniform in width. The projections studied in the bi-layer are clearly observed to be knob-like and consist of a head, a stalk and a base.
Two-dimensional crystals of membrane-bound Na+,K(+)-ATPase were formed in acidic media and their qualities were investigated by electron cryo-microscopy as well as by conventional electron microscopy. At pH 4.8 in sodium citrate buffer, the best crystallization condition, more than 80% of membranes formed crystals. The high ratio allowed high-resolution images to be taken by electron cryo-microscopy. Image processing revealed that they had unique lattice constants (a = 108.7 A, b = 66.2 A, gamma = 104.2 degrees) and had few defects in the crystalline arrays. The reconstituted Fourier map of the ice-embedded crystal showed that there are two high contrast parts in one unit cell.
To investigate the interaction between a signal peptide and the lipid bilayer, two kinds of peptides, L8-M5 (L8 = MRL8PLAALG, M5 = KVFER) and L14-M5 (L14 = MRL14PLAALG), were examined in membranes composed of dioleoylphosphatidylcholine (DOPC). Peptides L8 and L14 are artificially designed signal sequences, and M5 is the N-terminal five residues of human lysozyme; L8 mediated effective secretion of human lysozyme in yeast, while L14 did not [Yamamoto, Y., et al. (1987) Biochem. Biophys. Res. Commun. 149, 431-436]. DOPC liposomes incorporating L8-M5 or L14-M5 were observed by electron cryomicroscopy as pairs of concentric circles, and the separation of the bilayer was measured along the membrane. Peptide L8-M5 was found to reduce the bilayer thickness, but L14-M5 did not. CD measurements revealed that L8-M5 adopted an alpha-helical conformation with random coil in the liposome membranes and that L14-M5 adopted a more helical and less random conformation than L8-M5. Fluorescence spectroscopy using both aqueous and membranous probes revealed that L8-M5 destabilized the lipid bilayer more strongly than L14-M5. These results suggest that functional L8-M5 reduces the bilayer thickness and destabilizes the lipid bilayer and that these activities are important for signal peptide function.
Cross-sectional views of intact biomembranes and synthetic lipid bilayer membranes were observed by electron cryomicroscopy using spontaneous orientation of disk-shaped membranes; purple membrane, thylakoid membrane, synthetic phospholipid membrane and a microcrystalline sheet made of Er-binding polypeptide were observed. The membranes were observed fully hydrated, embedded in vitreous ice, and their self-orientation was most probably caused by repulsion between their hydrophobic edges and their hydrophilic environment which forced their edges to be exposed to the air-water interface. The cross-sectional profiles of the native biomembranes were asymmetric and characteristic, whilst those of the synthetic membranes were symmetric and predictable by a simple model. Simple Fourier analysis showed that the cross-sectional images retained structural information up to a medium resolution.
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The autopsy of a stillborn infant showed an extensive intravascular proliferation of atypical cells throughout the body. There was no infiltration by these cells of the parenchyma of the bone marrow, lymph nodes, or thymus. By histochemistry, these cells were negative for naphthol-ASD-chloroacetate esterase, a marker of granulocytes. By immunohistochemistry, they were reactive with MT-1 and anti-Leu-22, but unreactive with MB-1, L26, anti-Leu-M1, Tü-9, and antihemoglobin antibodies. A few cells reacted with antileukocyte common antigen and UCHL-1. Based on these findings, the lesion was diagnosed as angiotropic lymphoma (intravascular lymphomatosis) of the T-cell type, which occurred congenitally. Most angiotropic lymphomas in the literature are of the B-cell type, and no leukemia virus type I antibody was negative in the mother.
The irradiation damage of the crystals of proteins as well as nucleic acid is reduced almost exponentially even below the liquid nitrogen temperature down to 8K provided that the specimen area under illumination is cooled down. A cryo-stage cooled by superfluid helium is designed for the high resolution electron microscope based on the idea of the helium evaporation refrigerator with a capillary which was developed by Delong et al. We also developed a new top-entry-type cryo-transfer system for this superfluid cryo-electron microscope. The images of chlorinated Cu-phthalocyanine are taken to estimate the resolution attainable by this cryo-stage at 1.5 K with accelerating voltage of 400 kV. The optical diffraction confirms the resolution of 0.26 nm. The complexes of rec A proteins and DNA molecule are clearly visible in vitreous ice. A new membrane structure of the influenza virus is observed in the case of influenza A virus.