Proteoglycan arrays in the cochlear basement membrane.

Indirect immunofluorescence and transmission electron microscopy were used to investigate the composition and assembly of proteoglycans in the basement membranes of the spiral limbus, basilar membrane, spiral ligament, Reissner's membrane, myelinated nerve fibers, and blood capillaries of the spiral ligament and stria vascularis in the chinchilla cochlea. Four types of basement membrane components: laminin, entactin/nidogen, type IV collagen and heparan sulfate proteoglycans were immunolocalized in all basement membranes in association with heparan sulfate proteoglycans. beta 1 and alpha 1 integrin subunits were also detected along these basement membranes. The concentration of the basement membrane-associated proteins and integrin subunits differed according to the adjacent cell type. Electron microscopy showed that all basement membranes, with exception of those of stria vascularis, consist of two layers: lamina lucida and lamina densa. In the stria vascularis only a homogeneous lamina densa was observed. Cuprolinic blue treatment revealed heterogeneity in the ultrastructure and arrangement of proteoglycans in the cochlear basement membranes. Proteoglycans of the subepithelial basement membrane in the spiral limbus and spiral ligament formed quasi-regular, linear arrays within the lamina lucida, or were located at both sides of the lamina densa in the basilar membrane and Reissner's membrane. In the basement membranes of nerve fibers, and capillaries in the spiral ligament and stria vascularis, proteoglycans were scattered throughout these basement membranes, but showed different concentration and ultrastructural appearance, which may be related to different filtration and mechanical properties. In the basilar membrane, PGs were located above and below the lamina densa. An additional layer of PGs below the lamina densa may function as increased mechanical support of organ of Corti by its interaction with underlying fibrillar collagen layer. In the stria vascularis capillaries, PGs were stained considerably less with Cuprolinic blue and were scattered through the lamina densa of the basement membrane compared to capillaries of spiral ligament. This observation is compatible with a higher permeability of the strial capillaries.

Characterization of mucins in human middle ear and Eustachian tube.

Mucins are important glycoproteins in the mucociliary transport system of the middle ear and Eustachian tube. Little is known about mucin expression within this system under physiological and pathological conditions. This study demonstrated the expression of MUC5B, MUC5AC, MUC4, and MUC1 in the human Eustachian tube, whereas only MUC5B mucin expression was demonstrated in noninflamed middle ears. MUC5B and MUC4 mucin genes were upregulated 4.2- and 6-fold, respectively, in middle ears with chronic otitis media (COM) or mucoid otitis media (MOM). This upregulation of mucin genes was accompanied by an increase of MUC5B- and MUC4-producing cells in the middle ear mucosa. Electron microscopy of the secretions from COM and MOM showed the presence of chainlike polymeric mucin. These data indicate that the epithelium of the middle ear and Eustachian tube expresses distinct mucin profiles and that MUC5B and MUC4 mucins are highly produced and secreted in the diseased middle ear. These mucins may form thick mucous effusion in the middle ear cavity and compromise the function of the middle ear.

Helical structure of hair cell stereocilia tip links in the chinchilla cochlea.

Outer-hair-cell stereocilia tip-link structure in the chinchilla cochlea was studied by transmission electron microscopy using tannic acid and Ruthenium red/ Alcian blue histochemical procedures. Tannic acid and Ruthenium red/Alcian blue treatments showed the tip link as a compact strand of filaments 9-12 nm thick. Fourier analysis of tip-link images showed that the strand is a three-start helical bundle of fine, coiled filaments which had an axial period of 22.5+/-1.5 nm. Each of three coiled filaments in the strand showed globular structures, 4.3+/-0.3 nm in diameter. The globular structures may correspond to individual protein subunits or they may be repeating identical domains of one polypeptide. The three filaments of the helical array may provide a rigidity to the tip link during stereocilia deflections. Alternatively, changes in the subunit or domain structure of each filament may result in a lengthening or shortening of the tip-link strand.

Oligomeric structure of bAE3 protein.

The "brain" form of the anion exchanger protein 3 (bAE3) has been purified to homogeneity from the rabbit kidney by differential centrifugation and immunoaffinity chromatography. A single protein band of approximately 165 kDa was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Monomers, dimers (a major component), and a higher oligomeric form (apparently tetramers) were found after oxidative cross-linking of purified bAE3. The largest form of bAE3 was separated from dimers and monomers by sucrose gradient centrifugation and was studied by transmission electron microscopy to confirm a tetrameric structure. Two main types of bAE3 images were detected, round (approximately 11-14 nm) and square-shaped (approximately 12 x 12 nm). Image analysis revealed fourfold rotational symmetry of both the round and square-shaped images, indicating that bAE3 consists of multiples of 4 subunits. We conclude that bAE3 in Triton X-100 solution is predominantly a mixture of dimers and tetramers with a smaller amount of monomers.

Ultrastructure and immunohistochemical identification of the extracellular matrix of the chinchilla cochlea.

The molecular composition and three-dimensional organization of the extracellular matrix (ECM) was studied by immunofluorescent microscopy, transmission and scanning electron microscopy in three connective tissue structures of the cochlea: the spiral limbus, basilar membrane and spiral ligament. Type II collagen, fibronectin, tenascin, chondroitin sulfate proteoglycans, alphav and beta1 integrins were immunolocalized in the ECM of these connective tissue structures. Electron micrographs showed a continuum of cross-striated collagen fibrils having a similar diameter and axial periodicity that spread from the spiral limbus via the basilar membrane and into the spiral ligament. Some of collagen fibrils were aggregated laterally into bundles. Bundle images, and their digital Fourier transformations, showed a major 67-nm axial D-repeat characteristic for collagen fibrils. Transmission electron microscopy showed numerous proteoglycans associated with the collagen fibrils. The spiral limbus, basilar membrane and spiral ligament demonstrated regional differences in molecular composition and structural organization of their ECM. The glycoproteins fibronectin, tenascin and alphav integrin were immunolocalized mainly in the basilar membrane. Collagen fibrils of the spiral limbus and spiral ligament did not appear to be strongly oriented. However, most of the collagen fibrils in the basilar membrane were arranged into radially directed bundles. Collagen fibrils in the basilar membrane were also surrounded by a homogeneous matrix, which was immunoreactive to fibronectin and tenascin antibodies. A more complete understanding of the composition and structural organization of the ECM in these connective tissue structures in the cochlea provides a foundation upon which micromechanical models of cochlear function can be constructed.

Structure of outer hair cell stereocilia links in the chinchilla.

The structure of side, tip, and "attachment" links of chinchilla outer hair cell (OHC) stereocilia was studied by transmission and scanning electron microscopy using tannic acid and Cuprolinic blue histochemical procedures. Tannic acid, which interacts with many different types of proteins and glycoproteins irrespective of their electrical charge, showed strong reactivity for the central area of the side links and weak reactivity for the marginal area of these links adjacent to the stereocilia membrane. Tannic acid treatment revealed the tip links as thin strands, about 5 nm thick. Attachment links were poorly visualized after tannic acid treatment and appeared as sparse filamentous strands at tips of the tallest OHC stereocilia. Cuprolinic blue, at a high critical electrolyte concentration, reacted with strongly negative, primarily sulfated, carbohydrate residues of glycoconjugate macromolecules. In contrast to the tannic acid treatment, the central portions of the OHC stereocilia side links were unstained after Cuprolinic blue treatment; however, membrane-associated ends of these links were darkly stained. The tip links showed a similar appearance as after tannic acid treatment; however, Cuprolinic blue revealed an electron-dense substructure at both ends of its insertion into the stereocilia. Cuprolinic blue reactive structures were also observed as attachment links only at the tips of the OHC stereocilia of the tallest row in each bundle. These structures formed a crown-like array around the tip of each stereocilium. Their primary function appears to be attachment of type B fibrils of the tectorial membrane to the tallest OHC stereocilia. Cuprolinic blue reactive structures of the side, tip, and attachment links appear to contain acidic, sulfated residues of proteoglycans or glycoproteins. These structures may function as connective elements between the stereocilia links and the hair cell cytoskeleton.

Ultrastructural organization of proteoglycans and fibrillar matrix of the tectorial membrane.

The molecular and supramolecular structure of the tectorial membrane (TM) was studied by transmission electron microscopy (TEM). Collagen (type A) fibrils in the TM were found associated with proteoglycans (PGs) and type B fibrils. Most PGs were orthogonally oriented and attached D-periodically to collagen fibrils. Computer averaged projections of PG particles and linear aggregates of PGs in crystalline arrays, stained with Cuprolinic blue, showed an elongated, electron-dense structure 50-65 nm in length and 10 nm in width. Image analysis of type B fibrils showed that they are constructed of globular domains arranged with a periodicity of 12-14 nm. Each globular domain contains two thin 'arms', extended in opposite directions, which contact the 'arms' of adjacent fibrils. Numerous type B fibrils were found between collagen fibrils. They are attached to adjacent collagen fibrils by the 'arms' of their globular domains. An association of type B fibrils and PGs with collagen seems to result in the local ordered arrangement of the TM matrix. A hypothetical model of the TM matrix supramolecular structure is presented.

Crystalline arrays of proteoglycan and collagen in the tectorial membrane.

Ultrastucture of the tectorial membrane in the chinchilla cochlea was studied by transmission electron microscopy using different fixatives and staining procedures. It was shown that the tectorial membrane is a highly structured matrix composed of collagen type A fibrils, noncollagenous type B fibrils and proteoglycan. The localization of type B fibrils surrounding bundles of parallel type A fibrils was observed. Staining of the tectorial membranes with the cationic dye Cuprolinic blue in a "critical electrolyte concentration" method revealed proteoglycan, D-periodically associated with collagen type A fibrils and orthogonal to them. The appearance and size of the proteoglycan, and its binding to collagen, were similar to small proteoglycans observed in cartilage and other tissues. In many regions of the tectorial membrane the collagen-bound proteoglycan forms crystalline-like arrays. The images of these arrays processed by Fourier analysis show long linear aggregates of proteoglycan arranged parallel each other.

Electron microscopy of Bacillus subtilis GroESL chaperonin and interaction with the bacteriophage phi 29 head-tail connector.

The Bacillus subtilis GroESL chaperonin was isolated by sucrose density gradient centrifugation and the constituent GroES and GroEL moieties were purified by electrophoresis in agarose. Electron microscopic images of negatively stained GroEL and GroES oligomers and GroESL complexes were averaged using a reference-free alignment method. The GroEL and GroES particles had the sevenfold symmetry characteristic of their Escherichia coli counterparts. GroESL complexes, reconstituted efficiently in vitro from GroEL and GroES in the absence of added ADP or ATP, had the characteristic bullet- and football-like shapes in side view. Purified bacteriophage phi 29 head-tail connectors having a mass in excess of 0.4 MDa were shown to bind to GroESL at the end opposite to the GroES. The same GroESL-connector complexes were isolated from phage-infected cells in which capsid assembly was blocked, and thus the complex may have functional significance in phi 29 morphogenesis.

The bacteriophage phi 29 head-tail connector shows 13-fold symmetry in both hexagonally packed arrays and as single particles.

The symmetry of the phi 29 head-tail connector is controversial: several studies of two-dimensional arrays of the connector have found a 12-fold symmetry, while a recent study of isolated particles has found a 13-fold symmetry. To investigate whether a polymorphism of the structure might explain these different results, electron microscopy and image analysis were used to study both isolated connectors and particles in hexagonally packed arrays. The hexagonally packed arrays have a P1 symmetry, and the connectors displayed 13 subunits both in the arrays and as isolated single particles. While we do not observe a polymorphism between connectors in two-dimensional arrays and as isolated particles, data show that the connectors can exist with either 12 or 13 subunits. A three-dimensional reconstruction of our 13-fold connector was generated by combining an averaged side-view projection with the known symmetry. The structure of rosettes of the connectors formed in the presence of phi 29 prohead RNA (pRNA) was also examined. These rosettes contain five connectors arranged about a single connector in the center, and this arrangement may reflect an essential role of the pRNA in mediating a symmetry mismatch between either a 12- or 13-fold symmetric connector and a putative fivefold symmetric prohead portal vertex into which the connector fits.

[Some biochemical characteristics of substances from extracellular virus-like particles of the Gypsy retrotransposon (MDG4)]. / Issledovanie nekotorykh biokhimicheskikh kharkteristik preparatov vnekletochnykh virusopodobnykh chastits retrotranspozona Gypsy (Mdg4).

To investigate the gypsy functional activity, the possibility of conservation of its genetic information in VLP in the extracellular form has been examined. Preparations containing extracellular gypsy VLP from D. melanogaster and D. virilis were obtained. Non degradative full-sized polyA+ RNA and polyA+ RNA-DNA complexes of gypsy were revealed in the both preparations. The polypeptides with some specificity to gypsy nucleic acids were identified in VLP preparations. Some morphological and physical characteristics of the VLP preparations were obtained. The data obtained suggest the presence of non-degradative gypsy VLP in cultured media of D. melanogaster and D. virilis cells.

Trimeric forms of the photosystem I reaction center complex pre-exist in the membranes of the cyanobacterium Spirulina platensis.

Oligomeric and monomeric forms of chlorophyll-protein complexes of photosystem I (PSI) have been isolated from the mesophilic cyanobacterium Spirulina [(1992) FEBS Lett. 309, 340-342]. Electron microscopic analysis of the complexes showed that the oligomeric form is a trimer of the shape and dimensions similar to those of the trimer from thermophilic cyanobacteria. The chlorophyl ratio in the isolated trimer and monomer was found to be 7:3. The trimeric form of PSI complex in contrast to the monomeric one contains the chlorophyll emitting at 760 nm (77K), which is also found in Spirulina membranes and therefore could be used as an intrinsic probe for the trimeric complex. The 77K circular dichroism spectrum of the trimeric form is much more similar to that of Spirulina membranes than the spectrum of the monomer. Thus, the trimeric PSI complexes exist and dominate in the Spirulina membranes.

Cytochrome P-450: hexameric structure of the purified LM4 form.

Purified cytochrome P-450LM4 was found to be monodisperse in 20% glycerol by analytical ultracentrifugation. Its S20,w value was quite similar to that of hexameric P-450LM2. At lower glycerol concentrations the P-450LM4 showed a tendency to aggregate. The P-450LM4 oligomers were immobilized on Ultrogel A4 under conditions allowing only one covalent link to the matrix per oligomer. In the presence of SDS, the oligomers dissociated leaving only 1/6th of the initial amount of bound protein on the matrix, suggesting that the purified P-450LM4 is a hexamer. This was confirmed by electron microscopy. The quaternary structure of the P-450LM4 was similar to that demonstrated earlier for P-450LM2.

Presence of the gypsy (MDG4) retrotransposon in extracellular virus-like particles.

As a first step to investigate the functional activity of gypsy virus-like particles (VLPs) we explored the possibility of preservation of its VLP in extracellular form. The preparations containing extracellular gypsy VLP from Drosophila melanogaster and D. virilis were obtained. Full-length polyA+ RNA and polyA+ RNA-DNA complexes of gypsy were observed in both preparations. The polypeptides with some specificity to gypsy nucleic acids were identified in the obtained VLP preparations. These data accompanied by morphological characteristics of samples testify the presence of intact gypsy VLP in cultured media both from D. melanogaster and D. virilis cultivated cells.

[Analysis of the quaternary structure of secreted repressible acid phosphatase from the yeast Saccharomyces cerevisiae]. / Analiz chetvertichnoi struktury sekretiruemoi repressibel'noi kisloi fosfatazy drozhzhei Saccharomyces cerevisiae.

The structural organization of extracellular repressible acid phosphatase from S. cerevisiae has been studied. The existence of multiple acid phosphatase forms with isoelectric points at pH 4.1-4.8 has been confirmed by isoelectrofocusing. The molecular masses of three acid phosphatase isoforms (56, 57-59, and 60 kDa) obtained after enzymatic deglycosylation correlate with the data obtained previously during the analysis of translation products in cell-free systems. Electron microscopic studies revealed that the acid phosphatase molecule has a square shape and is made up of four identical subunits with molecular masses of about 125 kDa.

Isolation, crystallization in the macrogravitation field, preliminary X-ray investigation of uridine phosphorylase from Escherichia coli K-12.

Uridine phosphorylase (UPH) from Escherichia coli K-12 has been purified to near homogeneity from a strain harbouring the udp gene, encoding UPH, on a multicopy plasmid. UPH was purified to electrophoretic homogeneity with the specific activity 230 units/mg with a recovery of 80%, yielding 120 mg of enzyme from 3g cells. Crystals of enzyme suitable for X-ray diffraction analysis were obtained in a preparative ultracentrifuge. The packing of the molecules in the crystals may be described by the space group P2(1)2(1)2(1) with the unit cell constants a = 90.4; b = 128.8; c = 136.8 A. There is one molecule per asymmetric unit, Vm = 2.4. These crystals diffract to at least 2.5-2.7 A resolution. The hexameric structure of UPH was directly demonstrated by electron microscopy study and image processing.

Electron microscopy and image analysis of the GroEL-like protein and its complexes with glutamine synthetase from pea leaves.

The molecular structure of GroEL-like protein from pea leaves has been studied by electron microscopy and image analysis of negatively stained particles. Over 1500 molecular projections were selected and classified by multivariate statistical analysis. It was shown that the molecule consists of 14 subunits arranged in two layers with 72 point group symmetry. Side view projections of the molecule show a four-striation appearance, which subdivides both layers of seven subunits into two halves; this may be explained by a two-domain structure of the subunits. The presence in protein preparations of projections corresponding to one layer of subunits or half-molecules is consistent with the molecular structure suggested. Electron microscopic evidence for a specific association of GroEL-like protein and octameric glutamine synthetase, which was co-purified with this protein, was obtained.

The main adenosine triphosphate-binding component of amphibian oocytes is ferritin.

After fractionation of mitochondrion-free extracts of Xenopus laevis and Rana temporaria oocytes in sucrose gradients, a distinct peak of adenosine triphosphate (ATP)/guanosine triphosphate (GTP)-binding activity in the 50-70 S range has been detected. This substance has a boyant density in Cs2SO4 of 1.45 g/cm3. The nucleotide-binding substance has been purified to apparent homogenety. By means of electron microscopy, sodium dodecyl sulfate-electrophoresis and other methods it has been identified as ferritin.