Replica Analysis Research Articles

Morphology and Physiology of Gamete Mating and Gamete Fusion in the Fungus Allomyces

New structural features of gametes are presented and several physiological aspects of mating and gamete fusion in the fungus Allomyces macrogynus are described. In the posterior region of the male gametes, where fusion is thought to occur, thin-section electron microscopy demonstrated the presence of microfilaments that were associated with a ruffling of the otherwise smooth plasma membrane. When the cells were treated with the microfilament-disruptive drug cytochalasin B, fusion was inhibited due to the interaction of the drug with the female gamete. Freeze-fracture replica analysis of the posterior region of the gamete plasma membrane showed an area devoid of membrane particles (termed the flagellar collar) at the basal end of the flagellum. Physiological analysis of gamete mating and fusion showed a requirement for divalent cations, and the time required for gamete mating could be lengthened or shortened by decreasing or increasing, respectively, the divalent cation concentration. Diphenhydramine and chloroquine also increased the time required for mating or completely prevented fusion. Tertiary amine local anaesthetics produced the same type of results, as did trypsin and Pronase. Phospholipases were without an inhibitory effect. When the gametes were washed free of the drugs prior to mating, it was seen that the female gametes were more sensitive to cell surface changes than the males. In addition, 2-h-old female gametes showed a 70% loss of fusion ability with young male gametes while the reverse experiment showed only a 20% decrease. These observations support the concept that gamete mating and fusion may occur at the flagellar collar area and that mating in A. macrogynus is governed, at least in part, by cation and protein interactions.

Plaque sampling and telemetry for monitoring acid production on human buccal tooth surfaces

The plaque sampling method and wire telemetry using miniature, glass pH electrodes on the buccal surface of mandibular first permanent molars, compared human dental plaque pH responses to a 10 per cent solution of sucrose. Nine subjects abstained from oral hygiene and were tested in morning sessions on 5 consecutive days. The mean resting plaque pH value for all sessions by plaque sampling was 6.60 ± 0.18 (mean ± SD) and by telemetry was 6.56 ± 0.19. After a 1 min sucrose rinse, 1-day-old plaque showed a decrease in pH to approx. 5.5 by both methods. The 2-, 3-, 4- and 5-day-old plaque showed a consistent and more marked response to the carbohydrate. With 5-day-old plaque, the mean minimum pH achieved was 4.69 ± 0.28 as measured by sampling and 3.94 ± 0.49 as determined by telemetry. The times taken to reach minimum pH as determined by the two methods were almost identical. The electrode calibration data showed that all the telemetry electrodes responded consistently for all 5 days of study. Scanning electron microscopy and microbiological analysis of electrode tips and enamel replicas revealed that plaque accumulated on glass at the same rate and with a similar bacterial composition to plaque formed on clean enamel. It is proposed that glass microelectrodes and wire telemetry are a reasonable means for continuously monitoring plaque pH in situ. Comparison with plaque sampling suggested that telemetric responses reflect the type of plaque which accumulates at particular sites on the dentition.

Ultrastructural changes related to functional activity in gastric oxyntic cells.

When stimulated to secrete HCl the gastric oxyntic cell undergoes profound morphological change. The identifiable apical cell surface is greatly expanded in the stimulated oxyntic cell as compared with nonsecreting ones. To account for this change, one hypothesis proposes that the expanded surface is derived from the fusion of cytoplasmic tubulovesicular membranes with the existing limited apical membrane surface. An alternative hypothesis suggests that the tubulovesicular compartment is actually confluent with the apical surface at all times and that the morphological appearance follows the expansion of this supercollapsed compartment as HCl secretion commences. A variety of morphological evidence is reviewed here including transmission electron microscopy during various stages of secretion and inhibition, analysis of freeze-fracture replicas, penetration of macromolecular tracers, and membrane surface-staining characteristics. It is concluded that the weight of evidence favors a membrane fusion process. Moreover, recent comparative studies of membrane fractions from resting and secreting stomachs show different morphological and functional properties that are also consistent with a fusion hypothesis as a fundamental event in the membrane transformation of the oxyntic cell.

Morphology and vascular anatomy of the gills of a primitive air-breathing fish, the bowfin (Amia calva).

The morphology of the gills of a primitive air breather (Amia calva) was examined by light microscopy of semithin sections of gill filaments, and gill perfusion pathways were identified by scanning-electron microscopic analysis of corrosion replicas prepared by intravascular injection of methyl methacrylate. The arrangement of gill filaments and respiratory lamellae is similar to that ot teleosts with the exception of an interfilamental support bar that is fused to the outer margins of lamellae on adjacent filaments. The prebranchial vasculature is also similar to that of teleosts, whereas the postbranchial circulation of arches III and IV is modified to permit selective perfusion of the air bladder. Gill filaments contain three distinct vascular systems: (1) the respiratory circulation which receives the entire cardiac output and perfuses the secondary lamellae; (2) a nutrient system that arises from the postlammelar circulation and perfuses filamental tissues; (3) a network of unknown function consisting of subepithelial sinusoids surrounding afferent and efferent margins of the filament and traversing the filament beneath the interlamellar epithelium. Prelamellar arteriovenous anastomoses (AVAs) are rare, postlammelar AVAs are common especially at the base of the filament where they form a dense network of small tortuous vessels before coalescing into a large filamental nutrient artery. Unlike in most teleosts, the outer vascular margins of the lamellae are embedded in the interfilamental support bar and become the sole vasculature of this tissue. Arterial-arterial lamellar bypass vessels were not observed. Previously observed decreases in oxygen transfer across the gills during air breathing can be explained only by redistribution of blood flow between or within the respiratory lamellae.

Freeze-fracture analysis of gap junction disruption in rat ovarian granulosa cells

The effects of chemical dissociation on rat ovarian granulosa cell gap junctions has been studied using freeze-fracture electron microscopy. Sequential exposure of granulosa cells within follicles to solutions containing 6·8 mM EGTA [ethylene-bis-(β-aminoethyl ether)-N,N′-tetra acetic acid] and 0·5 M sucrose results in extensive cellular dissociation of the follicular epithelium. Freeze-fracture replicas made from fixed, control or EGTA-treated ovarian follicles exhibit extensive gap junctions between granulosa cells that are characterized by a range of packing order of constituent P-face particles or E-face pits. In contrast, exposure to 0·5 M sucrose containing 1·8 mM EGTA for as little as 1 min results in a consistently close packing of particles or pits which is accompanied by splitting of gap junctions between granulosa cells. The process of junction splitting was studied in detail in replicas prepared from follicles treated sequentially for various periods of time with EGTA and sucrose solutions. Initially, large gap junctions lose their regular shape and fragment into numerous tightly packed aggregates of P-face particles or E-face pits which are separated by unspecialized areas of plasma membrane. Subsequent to junction fragmentation, individual junction plaques separate at sites of cell contact and generate hemijunctions that border the intercellular space, Hemijunctions undergo particle dispersion of the P fracture face which results in an increased density of large intramembrane particles; no corresponding change in E-face pits is discernible at this stage. Morphometric analysis of replicas of tissue undergoing junction splitting indicates that junctional surface area decreases to 10–20% of control levels during this same treatment and so further supports the qualitative observations on junction fragmentation. Viabilities of granulosa cells obtained by these techniques also agree with the sequence observed in the morphometric analysis of the replicas. Finally, within 15 min after placing ovaries in isotonic, Ca 2+-containing salt solutions, gap junction reformation occurs by aggregation of particles at sites of intercellular contact. These sites are distinguished by the appearance of short surface protrusions or indentations on their respective P and E fracture faces. The data suggest a mechanism for EGTA-sucrose mediated cellular dissociation in the follicular epithelium in which gap junctional particles are free to move in the plane of the plasma membrane and may be re-utilized to form gap junctions in the presence of extracellular calcium.

Subunits of the triadic junction in fast skeletal muscle as revealed by freeze-fracture

Freeze-fracture analysis of triadic junctions utilizing an abbreviated glycerination schedule reveals regularly arranged intramembranous subunits within junctional portions of sarcoplasmic reticulum terminal cisterns. The subunits appear as round to square 20-nm elevations of the junctional terminal cistern E-face; the complementary P-face is characterized by a concentration of small particles, the detailed arrangement of which is less obvious. The E-face subunits may contain a pore, and their spatial arrangement corresponds to the tetragonal array of dimples or feet previously described within the junctional gap. No corresponding subunits have been detected within T-tubular membranes. Stereo techniques and complementary replica analysis have been utilized for detailed study of the subunits as well as associated features of the fracture faces of both T-tubular and terminal cistern junctional and nonjunctional membranes. The new findings are discussed in relation to structural models which might explain the mechanisms of the triadic junction involved in excitation-contraction coupling.

Quantitative ultrastructural analysis in cardiac membrane physiology

Quantitative measurements on electron micrographs of heart muscle can yield information useful for cellular physiologists and at present not obtainable in other ways. These methods are subject to preparative artifact, sampling problems, and problems inherent in the mathematical description of ultrastructure. Nevertheless they provide the best available data for membrane areas of the plasmalemma and its components, as well as for membrane areas of the sarcoplasmic reticulum and mitochondria. Morphometric methods can be used to study growth of membranes. Changes in the volumes of intracellular membrane-limited subcompartments can also be measured. Quantitative analysis of freeze-fractured membrane replicas can be carried out either by a statistical approach or by optical diffraction. In this way, physiological perturbations or developmental events leading to changes in membrane permeability can be studied for correlated changes in membrane structure.

The ependyma and subependymal layer of the young rat: A new contribution with freeze-fracture

The subependymal layer underlying the external wall of the lateral ventricle has been studied with freeze-fracture in 3-week-old rats. Membrane specializations corresponding to gap junction and attachment plates were found on ependymal cells, as well as the characteristic features of cilia and microvilli. Hexagonal arrays of particles previously found in the membrane of astrocytes were also present in ependyma. The membranes of subependymal cells were characterized by a paucity of intramembranous particles, when compared to ependymal and other mature glial cells. Specialized regions corresponding to areas of close membrane apposition seen on the sections were tentatively identified as clustering of particles on the protoplasmic face, corresponding with a cobbly texture on the exoplasmic face. Their possible significance, if they are not artifactual, is discussed. The analysis of replicas obtained from freeze-fractured material provides an additional tool for the study of cell differentiation and maturation, in our case gliogenesis. The comparison of membrane characteristics of neuroepithelial cells—precursors of glia—with those of mature glial cells and intermediate forms may contribute to a better understanding of the mechanism of their maturation.

Synthesis of dispersion-strengthened alloys by the activated reactive evaporation process from a single rod-fed electron beam source

Oxide-dispersion-strengthened alloys have better high-temperature properties but poorer low-temperature strength compared to Ni-base super alloys. Relative to these alloys, dispersion-strengthened alloys of titanium carbide dispersed in a Ni-base alloy matrix have been found to have good low- as well as high-temperature strength. This investigation was concerned with the synthesis of dispersion-strengthened alloys of TiC particles in a Ni matrix using the activated reactive evaporation (ARE) process. A Ni–Ti alloy rod was evaporated from an electron beam source in the presence of C2H2, all of the vapor species being activated or partially ionized using the ARE process. The deposits were characterized by x-ray diffraction, electron microprobe analysis, microhardness, and transmission electron miscroscopy of carbon replicas. It was established that Ni–TiC dispersion-strengthened alloys were produced by ARE process. The microhardness of the alloys decreased when annealed at high temperatures due to particle coarsening.

Unique cleavage planes in frozen red cell membranes.

SummaryThe Bullivant freeze-cleave technique has been modified to allow for the casting of replicas of the faces produced both above and below a single cleavage through frozen specimens. Analysis of replicas of paired fracture faces indicates that membrane faces normally revealed by cleaving are complementary and originate from the interior of the cell membrane. The thickness of the layers demonstrated by cleaving and heat-etching indicates that the potential cleavage plane from which membrane Face A and B are generated exists near the center of the membrane. The results provide physical evidence for the presence of a lipid bilayer within at least part of the red cell membrane.