A study of staining for electron microscopy using collagen as a model system—VIII. Simulation of the negative staining pattern

Simulated negative staining patterns of collagen fibrils were prepared for visual display by a graphical procedure in which amino acid side-chains along the staggered molecules were weighted according to their stain-excluding capacity. The simulated patterns were then compared directly with electron-optical images of collagen fibrils negatively stained with sodium phosphotungstate or lithium tungstate. These visual comparisons confirm previous observations that satisfactory matching occurs when side-chains are weighted according to their ‘bulkiness’ (average cross-sectional area or ‘plumpness’). Optimal matching at the edges of the overlap zones occurred when a hairpin-like conformation was assumed for the N-terminal telopeptides and a condensed conformation for the hydrophobic part of the C-terminal telopeptides. The negative staining pattern is known to include some element of positive staining; visual matching suggests that this additional uptake of positive staining ions occurs predominantly in the more accessible gap zone in a fibril D-period. A slight mismatching between observed and simulated patterns can be understood if the gap zone suffers greater axial shrinkage than the overlap zone when specimens are prepared for electron microscopy.     

Ultrastructure of meiosis in the hollyhock rust fungus,Puccinia malvacearum I. Prophase I — Prometaphase I

Summary A thoroughly documented account of the ultrastructure of the meiotic spindle pole body (SPB) cycle in a rust (Basidiomycota, Uredinales) is presented for the first time. The three-dimensional structure of the SPB and spindle during meiosis in the hollyhock rust fungusPuccinia malvacearum is analyzed from serial sections of preselected stages. This paper covers prophase I to prometaphase I. At late prophase I, the nucleolus disperses and does not reappear until the end of meiosis. The SPB at late prophase I consists of two, 4-layered discs, 0.8–1.0 m in diameter, connected by a middle piece (MP). The SPB is associated with a differentiated region of the nuclear envelope and nucleoplasm. At late diplotene to diakinesis, each disc generates a half spindle as it inserts into an otherwise intact nuclear envelope. The MP connecting the interdigitating half spindles elongates and eventually splits transversely during subsequent spindle elongation. Each half MP, which is attached to a SPB disc, becomes inserted in a sheath-like extension of the nuclear envelope. The intranuclear late prometaphase I spindle always becomes oriented perpendicularly to the longitudinal axis and sagittal plane of the metabasidium. There are 200–290 spindle microtubules (MTs) at each SPB at late prometaphase. The nonkinetochore MTs form a coherent central spindle around which the kinetochore MTs and bivalents are spread. A metaphase plate is absent. The results are compared with SPB behavior and spindle structure in early meiosis of other basidiomycetes and ascomycetes.     

A study of staining for electron microscopy using collagen as a model system—V. The effect of suberimidate fixation on negative staining patterns

The effects of the fixative dimethylsuberimidate (DMS) on negative staining patterns were studied using reconstituted fibrils of type I calf skin collagen (of known amino acid sequence) as a model system and comparing electron-optical data and chemical data by a computer-aided correlation procedure. The results show that the ‘bulkiness’ (average cross-sectional area or ‘plumpness’) of amino acid side chains is the dominant factor in determining the stain-excluding property of a DMS-fixed negatively stained collagen fibril as it is in unfixed collagen. Some contribution of positive staining can also be demonstrated after DMS-fixation by partial correlation analysis. Other evidence suggests that (unlike glutaraldehyde) DMS does not produce any morphological alterations to the negative staining pattern.     

Molecular Phylogenetic Relatedness of Frenkelia spp. (Protozoa,Apicomplexa) to Sarcocystis falcatula Stiles 1893: Is the Genus Sarcocystis Paraphyletic?

ABSTRACT The coccidians Frenkelia microti and F. glareoli (Apicomplexa: Sarcocystidae) form tissue cysts in the brain of small rodents (intermediate hosts) while oocysts are formed in the intestine of final hosts, buzzards of the genus Buteo. The inclusion of the small subunit ribosomal RNA gene sequences (SSU rRNA) of both Frenkelia species into the SSU rRNA trees of other, tissue cyst-forming coccidia strongly supports paraphyly of the genus Sarcocystis. Frenkelia spp. exhibit close relatedness to Sarcocystis falcatula Stiles 1893, a bird-opossum parasite, recognized under its junior synonym S. neurona Dubey et al. 1991, as the causative agent of equine protozoan myeloencephalitis on the American continent. As the definition of the genus Frenkelia is based on a plesiomorphic character (affinity to the neural tissue) of supposedly low phylogenetic value, the synonymization of the genus Frenkelia with Sarcocystis is proposed. This renders the genus Sarcocystis monophyletic.     

Ultrastructure of meiosis in the hollyhock rust fungus,Puccinia malvacearum II. Metaphase I—Telophase I

Summary The three-dimensional structure of the spindle pole body (SPB) and meiotic spindle during early metaphase I through telophase I inPuccinia malvacearum is analyzed ultrastructurally from serial sections. During early metaphase I the spindle rotates from the perpendicular to a position oblique to the longitudinal axis and parallel to the sagittal plane of the cell. Tubular cisternae are present within the spindle at this stage. The half middle piece (MP) subtends a collateral disc (co-disc) which is inserted eccentrically within each SPB. The SPB, co-disc and half MP at opposite poles are in mirror image. During the transition from early metaphase I to full metaphase I, the spindle orients parallel to the lateral wall of the promycelium and the half MPs are lost. The co-discs partially detach from each discoid SPB and maintain this relation until the end of interphase I. Co-discs become further differentiated as they attach to the subtending sheath-like extension of the nuclear envelope previously occupied by the half MPs. Microvesicles within the nucleoplasm are specific to mid metaphase I. A metaphase plate is absent. The 14 bivalents, which are directly connected to each polar SPB by 2 to 3 kinetochore MTs, are spread over nearly the entire length of the central spindle. The first anaphasic movement involves asynchronous shortening of the kinetochore MTs while the second consists of extensive pole-to-pole elongation. Astral MTs first appear at early metaphase I and become most numerous at anaphase I. An intact nuclear envelope constricts against the central spindle at either end of the interzonal region. Concurrently, centripetal growth of the nuclear envelope under each SPB results in their gradual externalization by the end of telophase I. The sibling nuclei are cut off by constriction of the nuclear envelope at either end of the interzonal region. These meiotic stages inP. malvacearum are compared with those in other basidiomycetes and ascomycetes.     

A study of staining for electron microscopy using collagen as a model system—VI. Uranyl nitrate negative staining patterns and their correlation with sequence data

Collagen is used as a model system to study the mechanism of negative staining. Negative staining patterns from reconstituted fibrils of type I calf skin collagen (of known amino acid sequence) were compared with chemical data by a computer-aided correlation procedure. The stain used was uranyl nitrate, pH 3.2 and 4.9. The results show that the ‘bulkiness’ (average cross-sectional area or ‘plumpness’) of amino acid side chains is the dominant stain-excluding factor determining the small-scale distribution of stain along the collagen fibril. Some contribution of positive staining can also be demonstrated by the analysis described here.     

Quantification of Collagen Ultrastructure after Penetrating Keratoplasty – Implications for Corneal Biomechanics

Purpose

To quantify long-term changes in stromal collagen ultrastructure following penetrating keratoplasty (PK), and evaluate their possible implications for corneal biomechanics.

Methods

A pair of 16 mm post-mortem corneo-scleral buttons was obtained from a patient receiving bilateral penetrating keratoplasty 12 (left)/28 (right) years previously. Small-angle x-ray scattering quantified collagen fibril spacing, diameter and spatial order at 0.5 mm or 0.25 mm intervals along linear scans across the graft margin. Corresponding control data was collected from two corneo-scleral buttons with no history of refractive surgery. Wide-angle x-ray scattering quantified collagen fibril orientation at 0.25 mm (horizontal)×0.25 mm (vertical) intervals across both PK specimens. Quantification of orientation changes in the graft margin were verified by equivalent analysis of data from a 13 year post-operative right PK specimen obtained from a second patient in a previous study, and comparison made with new and published data from normal corneas.

Results

Marked changes to normal fibril alignment, in favour of tangentially oriented collagen, were observed around the entire graft margin in all PK specimens. The total number of meridional fibrils in the wound margin was observed to decrease by up to 40%, with the number of tangentially oriented fibrils increasing by up to 46%. As a result, in some locations the number of fibrils aligned parallel to the wound outnumbered those spanning it by up to five times. Localised increases in fibril spacing and diameter, with an accompanying reduction in matrix order, were also evident.

Conclusions

Abnormal collagen fibril size and spatial order within the PK graft margin are indicative of incomplete stromal wound remodelling and the long term persistence of fibrotic scar tissue. Lasting changes in collagen fibril orientation in and around PK wounds may alter corneal biomechanics and compromise the integrity of the graft-host interface in the long term.     

Membrane potential difference of isolated plant vacuoles: positive or negative? I. Evidence for membrane binding of cationic probes

The binding of membrane potential cationic probes was studied on phospholipidic liposomes by equilibrium dialysis and microelectrophoresis. Surface binding of lipophilic cations (benzyltributylammonium or tetraphenylphosphonium) appears to be the major accumulation mechanism in liposomes and simulates the existence of a negative transmembrane potential (E_m), in absence of any transmembrane ionic gradient. Furthermore, this apparent negative potential has a classical response with regard to common E_m effectors, namely a depolarization induced by KCl or FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone). The relevance of these results to the study of transtonoplast potential difference on isolated vacuoles was investigated. Tetraphenylphosphonium was shown to bind to the tonoplast, the essential features of binding and interaction with E_m effectors being similar in vacuoles and liposomes. Therefore the assumption of negligible binding of cationic probe to vacuoles, classically admitted in determinations of vacuolar E_m using lipophilic cations, is untenable.     

A study of staining for electron microscopy using collagen as a model system—IV. Phosphotungstate/tungstate negative staining patterns and their correlation with sequence data

The type I collagen fibril (for which the axial distribution of amino acid residues is known) has been used as a model system to study the mechanism of negative staining. Negative staining patterns from reconstituted fibrils were compared with chemical data by a computer-aided correlation procedure. Stains used were: phosphotungstic acid, pH 3.2 and 7.0, lithium tungstate, pH 7.2, and methylamine tungstate, pH 6.6. In all cases, the results of the correlation analyses point to the ‘bulkiness’ (average cross-sectional area or ‘plumpness’) of amino acid side chains as the dominant stain-excluding factor determining the small-scale distribution of stain along the collagen fibril. Some preferred uptake of heavy metal ions on charged side chains (a positive staining contribution) can be demonstrated by partial correlation analysis but, under the staining conditions used here, the effect is largely masked by the much greater negative staining component.     

Ultrastructure of the spore in four Japanese species of Ptychomitrium Fürnr. (Musci)

The ultrastructure of the mature spore in four Japanese species of the acrocarpous moss genus Ptychomitrium is presented. In all species the spores have a similar pattern: there is no recognisable aperture nor sporoderm polarity, exine and perine are poorly developed, cytoplasm only occasionally shows polarity, and plastids have a well developed inner membrane system. The presence of frequent intine protrusions is a remarkable feature of this genus. A multilaminar structure of the exine, already observed in Grimmia, occurs also in these species although here it is less pronounced. The significance of these features is discussed within these species, as well as in comparison to other taxa, especially the genus Grimmia.     

Dinoflagellate,Euglenid, or Cercomonad? The Ultrastructure and Molecular Phylogenetic Position of Protaspis grandis n. sp.

Protaspis is an enigmatic genus of marine phagotrophic biflagellates that have been tentatively classified with several different groups of eukaryotes, including dinoflagellates, euglenids, and cercomonads. This uncertainty led us to investigate the phylogenetic position of Protaspis grandis n. sp. with ultrastructural and small subunit (SSU) rDNA sequence data. Our results demonstrated that the cells were dorsoventrally flattened, shaped like elongated ovals with parallel lateral sides, 32.5-55.0 mum long and 20.0-35.0 mum wide. Moreover, two heterodynamic flagella emerged through funnels that were positioned subapically, each within a depression and separated by a distinctive protrusion. A complex multilayered wall surrounded the cell. Like dinoflagellates and euglenids, the nucleus contained permanently condensed chromosomes and a large nucleolus throughout the cell cycle. Pseudopodia containing numerous mitochondria with tubular cristae emerged from a ventral furrow through a longitudinal slit that was positioned posterior to the protrusion and flagellar apparatus. Batteries of extrusomes were present within the cytoplasm and had ejection sites through pores in the cell wall. The SSU rDNA phylogeny demonstrated a very close relationship between the benthic P. grandis n. sp. and the planktonic Cryothecomonas longipes. These ultrastructural and molecular phylogenetic data for Protaspis indicated that the current taxonomy of Protaspis and Crythecomonas is in need of re-evaluation. The composition and identity of Protaspis is reviewed and suggestions for future taxonomic changes are presented. Problems within the genus Cryothecomonas are highlighted as well, and the missing data needed to resolve ambiguities between the two genera are clarified.     

Some contributions of I.E. Purkyn? to the visual physiology

In the research of binocular vision J. E. Purkyn? pointed out the role of retinal corresponding areas. He observed binocular interaction of pressure phosphenes elicited in them and the fusion of images projected on the corresponding foveal areas even in eyes with artificial divergency. For the first time he proposed the examination of visual fields (perimetry) in several planes and he also introduced the colour perimetry. Purkyn? analyzed profoundly the different functions of direct (foveal) and indirect (peripheral) vision. In indirect vision he anticipated the modern concept of the useful field of view. Of fundamental importance was Purkyn?'s research of the strategy and different types of ocular movements. In analysis of motion perception, he evolved the hypothesis of "general sense of space" which is very close to modern feed-back conception of "corollary discharge".     

Ultrastructure of spermatozoa of Solifuges (Arachnida, Solifugae): possible characters for their phylogeny?

The ultrastructure of spermatozoa is a widely accepted source of characters for phylogenetic studies. In this study the fine structure of sperm cells of representatives of six different New and Old World families (Ammotrechidae, Daesiidae, Eremobatidae, Galeodidae, Karschiidae, Solpugidae) of solifuges (Arachnida, Solifugae) were investigated in order to reveal putative characters suitable for subsequent systematic and phylogenetic analyses. The spermatozoa of solifuges represent a relatively simple type of sperm cells. In general, their spermatozoa are roundish, oval shaped (Ammotrechidae, Daesiidae, Eremobatidae, Solpugidae) or plate-shaped (Karschiidae) with or without membrane protuberances and devoid of a flagellum. Only in Galeodidae, very conspicuous thin and elongated sperm cells occur. The spermatozoa either occur as single cells (Eremobatidae, Solpugidae) or in groups of loose knit cells (Ammotrechidae) or in highly ordered groups (Karschiidae). In contrast to the other families studied here, within the Galeodidae and in the genus Blossia (Daesiidae) sperm cells surrounded by a secretion sheath, clearly representing coenospermia, could be observed.     

A negative staining study of natural and synthetic L-α-lysophosphatidylcholine micelles,macromolecular aggregates and crystals

A comparative assessment has been made by transmission electron microscopy of negatively stained specimens, of the micellar, aggregated and crystalline states of palmitoyl, oleoyl and ex ovo L-α-lysophosphatidylcholine present in aqueous suspensions. Micelle formation from dry lysophosphatidylcholines is shown to be temperature dependent. The presence of the unsaturated fatty acid in oleoyl L-α-lysophosphatidylcholine and some degree of unsaturation in L-α-lysophosphatidylcholine (ex ovo) promotes micelle formation at low temperatures (4 and 22°C), whereas crystalline palmitoyl L-α-lysophosphatidylcholine is essentially insoluble at low temperatures and requires incubation at 60°C to produce a micellar suspension.It is suggested that the micellar conformation is not spherical, a cylindrical or discoid shape is more compatible with the images presented. Both palmitoyl and ex ovo L-α-lysophosphatidylcholine produce flexibel rod-like micellar aggregates ca 6 nm in diameter and larger (20–60 nm dia) stacked-disc aggregates, again with a temperature dependency. The thickness of the disc-like L-α-lysophosphatidylcholine of a phospholipid bilayer (ca 6–7 nm). This, together with the ability of palmitoyl L-α-lysophosphatidylcholine to crystallize as multi-lamellar hexagonal particles which remain stable in aqueous suspensions at 4°C, suggests that, as with other phospholipids, the L-α-lysophosphatidylcholines possess the property of forming lamellar structures, but that these become increasingly unstable at higher temperatures depending on the fatty acid unsaturation. Ammonium molybdate and sodium phosphotungstate have been found to be more satisfactory than uranyl acetate for negative staining of aqueous suspensions of L-α-lysophosphatidylcholines.     

β-Adrenoceptors in the Tree Shrew Brain. I. Distribution and Characterization of [125I]Iodocyanopindolol Binding Sites

1. The number and distribution pattern of -adrenergic receptors in the brain have been reported to be species specific. The aim of the present study was to describe binding of the -adrenoceptor ligand [¹²⁵I]iodocyanopindolol in the brain of the tree shrew (Tupaia belangeri), a species which provides an appropriate model for studies of psychosocial stress and its consequences on central nervous processes.2. ¹²⁵I-Iodocyanopindolol (¹²⁵ICYP) labeling revealed a high degree of nonspecific binding, which was due mainly to interactions of this ligand with serotonin binding sites. For a quantitative evaluation of ₁- and ₂-adrenoceptors, serotonin binding sites had to be blocked by 100 M 5HT.3. Binding of the radioligand to ₁- and ₂-adrenoceptors was characterized using the ₁-specific antagonist CGP20712A and the ₂-specific antagonist ICI118.551. ₁-adrenoceptor binding is present in the whole brain, revealing low receptor numbers in most brain regions (up to 1.5 to 2.7 fmol/mg). A slight enrichment was observed in cortical areas (lateral orbital cortex: 4.0±0.7 fmol/mg) and in the cerebellar molecular layer (8.7±1.0 fmol/mg).4. Competition experiments demonstrated high- and low-affinity binding sites with considerable variations in K _i values for CGP20712A, showing that various affinity states of ₁-adrenoceptors are present in the brain (K _i: 0.61 nM to 67.1 M). In the hippocampus, only low-affinity ₁-adrenoceptors were detected (K _i: 1.3±0.2 M). Since it is known that ¹²⁵ICYP labels not only membrane bound but also internalized -adrenoceptors, it can be assumed that the large population of the low-affinity sites represents internalized receptors which may be abundant due to a high sequestration rate.5. High numbers of ₂-adrenoceptors are present in only a few brain structures of tree shrews (external layer of the olfactory bulb, 15.8±2.0 fmol/mg; claustrum, 19.3±1.5 fmol/mg; anteroventral thalamic nucleus, 19.4±1.5 fmol/mg; cerebellar molecular layer, 55.0±4.3 fmol/mg). Also for this class of -adrenoceptors, high- and low-affinity binding sites for the ₂-selective antagonist ICI118.551 were observed, indicating that ¹²⁵ICYP labels membrane bound and internalized ₂-adrenoceptors. Only in the cerebellar molecular layer was a high percentage of high-affinity ₂-adrenoceptors detected (K _i for ICI118.551 was 1.8±0.3 nM for 90% of the receptors).6. In conclusion, ₁- and ₂-adrenoceptor binding can be localized and quantified by in vitro receptor autoradiography in the brains of tree shrews when serotonergic binding sites are blocked. Modulatory effects of long-term psychosocial conflict on the central nervous -adrenoceptor system in male tree shrews are described in the following paper.