Tridimensional analysis of the formation of secretory vesicles in the Golgi apparatus of absorptive columnar cells of the mouse colon

The tridimensional structure of the Golgi apparatus has been studied in the absorptive cells of the mouse colon by means of reduced osmium postfixation and phosphatase cytochemistry. In thick sections of tissue impregnated with osmium tetroxide or treated with a technique to demonstrate TPPase activity, the Golgi formed a continuous ribbon-like structure capping the upper pole of the nucleus. Along the longitudinal axis of this ribbon, compact zones made up of superposed flattened saccules alternated with less compact zones which consisted of highly perforated saccules or bridging anastomosed tubules. In the cis-trans axis, the following elements were observed: (1) a cis element consisting of a continuous osmiophilic tubular network; (2) two or three subjacent elements selectively perforated by wells; (3) a trans compartment made up of two or three TPPase-reactive sacculotubular elements, some showing a "peeling-off" configuration. In some regions, the first flattened saccule of this trans compartment displayed discrete ovoid dilatations, located in compact zones and containing a dense granulofibrillar material; in the subjacent elements this material was seen concentrated in nodular swellings, at the intersection of the meshes of anastomosed membranous tubules. 100-300 nm vesicles containing a similar dense granulofilamentous material were observed in the trans Golgi zone and interspersed in the supranuclear cytoplasm between the Golgi zone and the apical surface of the cell. Smaller vesicles 80-100 nm in diameter containing a fine dusty material were also seen in proximity. These morphological observations suggested that at least two kinds of material were segregated in the saccules of the trans compartment and packaged in vesicles of two class sizes that detached from the Golgi stack on its trans aspect.     

Core substructure in Mastigocladus laminosus phycobilisomes

A native high molecular complex (M_r 850000) containing about 50% of the allphycocyanin of the phycobilisome but lacking allophycocyanin B was separated from isolated phycobilisomes by gel electrophoresis. It was designated AP_CM since the large linker polypeptide L_CM was exclusively localized in this complex. The complex exhibited a ?196°C fluorescence emission maximum at 673 nm (671 nm at 25°C). In addition, a core complex (designated AP_C, M_r≥1000000) consisting of both AP_CM and AP 680 was isolated by combined gel filtration and linear gradient centrifugation. At 25°C this complex showed dual emission peaks at 670 and 680 nm demonstrating functional independence of the terminal emitters. A complex similar to AP_CM can be isolated from phycobilisomes of Anabaena variabilis. This is evidence that AP_CM is the constitutive center of the tricylindrical core of hemidiscoidal cyanobacterial phycobilisomes. Two models summarizing the structural and functional consequences of the results are presented in the discussion.     

An 83-kDa embryonic-type nuclear antigen is detected within the germinal vesicles of oocytes of the ascidianHalocynthia roretzi

Summary Monoclonal antibodies were raised against germinal vesicles which were isolated from fully grown oocytes of the ascidianHalocynthia roretzi. Immunoblot analyses revealed that one of the antibodies, designated Hgv-2, recognized a single band with a molecular weight of about 83 kDa. The antibody, visualized by indirect immunohistochemistry, reacted only with the germinal vesicles of oocytes and did not react with test cells, follicle cells, and other somatic cells of the gonad. During embryogenesis the antigenicity was found in interphase nuclei of all embryonic cells. The antibody did not react with chromosomes or the mitotic apparatus. The antigenicity was retained by interphase nuclei of larval cells, but it disappeared from nuclei of juveniles about 7 days after metamorphosis.     

A re-evaluation of the cytology of cat Pacinian corpuscles

Summary The ultrastructure of cat mesenteric Pacinian corpuscles in cross and longitudinal sections has been examined. The terminal ends of lamellar cells of the inner core have been identified in longitudinal sections through the proximal portion of the inner core. These terminal bulbous expansions contain characteristic concentric membranes of rough endoplasmic reticulum and in some cases masses of oval membranous inclusions. The central axon as seen in cross section is oval in profile, having X-(short) and Y-(long) axes, and each axonal face is characterized by specializations of the axolemma. At the X-axis, the inner lamellae of the inner core tightly abut a smooth axolemma, with no intervening connective tissue matrix, in a manner reminiscent of a neuroepithelium. The axolemma of the Y-axis has numerous axonal spines (microspikes) that project into the cleft in the inner core. The extent of the axolemma having axonal spines can only be appreciated in longitudinal sections. The clefts contain a specialized connective tissue with elastic and collagen fibrils. The connective tissue compartment of fibers and matrix separating individual inner core lamellae is unique, in that it contains extremely thin collagen fibrils measuring approximately 15 nm in diameter. The diameter of collagen fibrils increases as the cleft is approached. Here the fibrils resemble typical endoneural collagen.     

A hemagglutinin specific for sialic acids in a rat brain synaptic vesicle-enriched fraction

A hemagglutinating activity was detected in a synaptic vesicle-enriched fraction prepared from adult rat brain, using trypsinized glutaraldehyde-fixed rabbit erythrocytes. The specific activity of the fraction, in two series of experiments, was 7.5 and 16-fold higher than in the other subcellular fractions. The activity was absent from the synaptosome cytosol. In a study using twenty-five different carbohydrates and glycoproteins, best inhibitors were N-acetylneuraminic acid and N-glycolylneuraminic acid, together with bovine submaxillary mucin and a glycopeptide fraction prepared from rabbit erythrocyte membranes. The activity was thermolabile and very sensitive to proteolytic enzymes (but insensitive to neuraminidase) indicating that a protein (agglutinin) is responsible for the activity. Experiments using detergents and high ionic strength showed that the agglutinin is tightly bound to membranes, inactivated by the so-called non denaturing detergents, and stable in diluted sodium dodecyl sulphate. Hypotheses are discussed on the possible function of the agglutinin.     

Structural and functional domains of cellobiohydrolase I from trichoderma reesei

Limited proteolysis (papain) of the cellobiohydrolase I (CBH I, 65 kDa) from Trichoderma reesei led to the seperation of two functional domains: a core protein (55 kDa) containing the active site, and a C-terminal glycopeptide (10 kDa) implicated in binding to the insoluble matrix (cellulose). The quaternary structures of the intact CBH I and its core in solution are now compared by small angle X-ray scattering (SAXS) measurements. The molecular parameters derived for the core (Rg=2.09 nm, D_max=6.5 nm) and for the intact enzyme (Rg=4.27 nm, D_max=18 nm) indicate very different shapes. The resulting models show a tadpole-like structure for the intact enzyme where the isotropic part coincides with the core protein and the flexible tail part should be identified with the C-terminal glycopeptide. Thus in this enzyme, functional differentiation is reflected in structural peculiarities.Abbreviations SAXS small angle X-ray scattering - SDS-PAGE SDS-polyacrylamide gel electrophoresis - IEF-PAG polyacrylamide gel isoelectric focusing; cellobiohydrolase (CBH, 1,4--glucan cellobio hydrolase (E.C.3.2.1.91)) - D_max maximum diameter - Rg radius of gyration     

Partial purification and characterization of a cellular protein that binds to the SV40 core origin of DNA replication

A monkey cell factor that interacts specifically with double- and single-stranded DNA sequences in the early domain of the simian virus 40 (SV40) core origin of replication was identified using gel-retention assays. The protein was enriched over 1200-fold using ion-exchange and affinity chromatography on single-strand DNA cellulose. Binding of protein to mutant origin DNA restriction fragments was correlated with replication activity of the mutant DNAs. Exonuclease footprint experiments on single-stranded DNA revealed prominent pause sites in the early domain of the core origin. The results suggest that this cellular protein may be involved in SV40 DNA replication.     

Effects of membrane potential on Na cotransports in eel intestinal brush-border membrane vesicles: Studies with a fluorescent dye

Summary The Na-dependent transport of a number of organic molecules (d-glucose,l-proline,l-alanine,l-phenylalanine) in brush-border membrane vesicles isolated from the intestine of the eel (Anguilla anguilla) was monitored by recording the fluorescence quenching of the voltage-sensitive cyanine dye 3,3-diethylthiacarbocyanine iodide (DiS-C₂(5)). The experimental approach consisted of: a) generating an inside-negative membrane potential mimicking in vivo conditions: b) measuring the rate of membrane potential decay (i.e., the rate of fluorescence quenching decay) due to Na-neutral substrate cotransport. Rates of membrane potential decay showed saturation on substrate concentration andK _app values (the substrate concentration giving 50% of the maximal rate) were estimated for Na-dependent transport ofd-glucose (0,099mm),l-alanine (0.516mm),l-proline (0.118mm) andl-phenylalanine (2.04mm). The influence of an inside-negative membrane potential on the affinity of the transporter for glucose and for sodium is discussed.     

The plasma membrane and generative cell organization in pollen of the mimosoid legume,Acacia retinodes

Summary Cytological changes associated with the final maturation, and dehiscence of the 16-grain compound pollen (polyads) have been followed in anthers at female and male phase of flowering. InAcacia retinodes, the transition from female to male phase takes approximately 24 h. The spherical generative cell at female phase is connected with the vegetative cell plasma membrane by a junction zone. This is sited adjacent to a germinal aperture, comprising wall ingrowths and membrane labyrinths. By male phase, the generative cell has elongated into a spindle-shape, and its surface is characteristically scalloped. The membrane labyrinths, especially those at the apertures, now contain masses of coated vesicles, implicated in the transport and secretion of proteins. Two-dimensional projections indicate that the generative cell and vegetative nucleus are closely associated forming a male germ unit.     

Freeze-substitution improves the ultrastructural preservation of legume root hairs

The freeze-substitution method was applied toVicia hirsuta root hairs to test its effectiveness in improving preservation of the cell ultrastruture. Freeze-substitution almost certainly represents more faithfully the structure of the hair cell. A previously unreported ‘pyriformis’ vesicle is described. Also unique to freeze-substituted material are coated secretory vesicles; a smooth plasma membrane profile; mitochondrial ribosomes; long microfilament bundles which are associated with vesicles, mitochondria, coated pits and coated vesicles; microtubule-associated filaments; well-preserved coated vesicles and coated pits with enclosing rings; a pliciform nucleus. The results are discussed in context of previous reports using conventional fixation techniques.