Rotary replication for freeze-etching.

Rotary replication has been adapted to freeze-etching and evaluated using T4 polyheads, erythrocyte ghosts, and chloroplast membranes. Conventional electron microscopy, electron diffraction, and optical diffraction and filtering indicate that platinum-carbon rotary replication renders radially symmetrical contrast and 25 A resolution to freeze-etched specimens so as to clarify subunit structure not normally evident in unidirectional shadow replicas.

Effects of pre- and postnatal exposure to 1880-1900MHz DECT base radiation on development in the rat.

In the present study, to evaluate the effects of wireless 1880-1900MHz Digital Enhanced Communication Telephony (DECT) base radiation on fetal and postnatal development, Wistar rats were exposed at an average electric field intensity of 3.7V/m, 12h/day, during pregnancy. After parturition, a group of dams and offspring were similarly exposed for another 22days. Controls were sham-exposed. The data showed that DECT base radiation exposure caused heart rate increase in the embryos on the 17th day of pregnancy. Moreover, significant changes on the newborns' somatometric characteristics were noticed. Pyramidal cell loss and glia fibrilliary acidic protein (GFAP) over-expression were detected in the CA4 region of the hippocampus of the 22-day old pups that were irradiated either during prenatal life or both pre- and postnatally. Changes in the integrity of the brain in the 22-day old pups could potentially be related to developmental behavioral changes during the fetal period.

Isolation and Characterization of Sex-Linked Female-Sterile Mutants in DROSOPHILA MELANOGASTER with Special Attention to Eggshell Mutants.

To study genes that function mainly or exclusively during oogenesis, we have isolated and analyzed female-sterile mutations, with special emphasis on those that affect eggshell formation. Following treatment that induced 61 to 66% lethals, 8.1% of the 1071 X chromosomes tested carried recessive female sterility mutations (87 isolates), and 8.0% carried partial female-sterile mutations (86 isolates), respectively. In addition, three dominant female steriles were recovered. Some of the mutants had very low fecundity, and others laid morphologically normal eggs that failed to develop. A third category included 29 mutants that laid eggs with morphological abnormalities: 26 were female steriles, two were partial female steriles and one was fertile. Mutants of this third category were characterized in some detail and compared with 40 previously isolated mutants that laid similarly abnormal eggs. Approximately 28-31 complementation groups with morphological abnormalities were detected, some of which were large allelic series (11, 9, 7, 6 and 5 alleles). Twenty-four groups were mapped genetically or cytogenetically, and 21 were partially characterized by ultrastructural and biochemical procedures. Of the latter, one group showed clear deficiency of yolk proteins, and nine showed prominent ultrastructural defects in the chorion (at least eight accompanied by deficiencies in characterized chorion proteins). At least six groups with clear-cut effects were found at loci not previously identified with known chorion structural genes.

Differential sorting of constitutively co-secreted proteins in the ovarian follicle cells of Drosophila.

Conventional and freeze-fracture electron microscopy, immuno-electron microscopy of ovarian cryosections and confocal immunofluorescence were used to analyze the ovarian distribution of the major protein classes being secreted by the follicle cells during the vitellogenic and choriogenic stages of Drosophila oogenesis. Our results clearly demonstrated that at vitellogenic stages the follicle cells co-secrete constitutively vitelline membrane and yolk proteins that are either sorted into distinct secretory vesicles or they are segregated in different parts of bipartite vesicles by differential condensation. Following their exocytosis only the vitelline membrane proteins are incorporated into the forming vitelline membrane. The yolk proteins (along with their hemolymph circulating counterparts) diffuse through gaps amongst the incomplete vitelline membrane and are internalized through endocytosis by the oocyte where they are finally stored into modified lysosomes referred to as alpha-yolk granules. The unexpected immunolocalization of vitelline membrane antigens in the associated body of the alpha-yolk granules may indicate that this structure is a transient repository for the proteins being internalized into the oocyte along with the yolk proteins. In the early choriogenic follicle cells the vitelline membrane and early chorion proteins were found to be co-secreted and to be evenly intermixed into the same secretory vesicles. These findings illuminate new details concerning the follicle cells secretory and oocyte endocytic pathways and provide for the first time evidence for condensation-mediated sorting of constitutively secreted proteins in Drosophila.

Stage-specific apoptotic patterns during Drosophila oogenesis.

In the present study we demonstrate the existence of two apoptotic patterns in Drosophila nurse cells during oogenesis. One is developmentally regulated and normally occurs at stage 12 and the other is stage-specific and is sporadically observed at stages 7 and 8 of abnormally developed follicles. The apoptotic manifestation of the first pattern begins at stage 11 and is marked by a perinuclear rearrangement of the actin cytoskeleton and the development of extensive lobes and engulfments of the nurse cell nuclei located proximal to the oocyte. Consequently, at late stage 12 (12C), half of the nurse cell nuclei exhibit condensed chromatin, while at late stage 13 all the nuclei have fragmented DNA, as it is clearly shown by TUNEL assay. Finally, the apoptotic vesicles that are formed during stage 13, are phagocytosed by the neighboring follicle cells and at stage 14 the nurse cell nuclear remnants can be easily detected within the adjacent follicle cell phagosomes. In the second sporadic apoptotic pattern, all the nurse cell nuclei are highly condensed with fragmented DNA, accompanied by a completely disorganized actin cytoskeleton. When we induced apoptosis in Drosophila follicles through an etoposide and staurosporine in vitro treatment, we observed a similar pattern of stage-specific cell death at stages 7 and 8. These observations suggest a possible protective mechanism throughout Drosophila oogenesis that results in apoptosis of abnormal, damaged or spontaneously mutated follicles before they reach maturity.

Effects of gamma rays on the stability and size of DNA.

The effects of gamma radiation on the stability and size of mammalian DNA were studied by using thermal transition spectrophotometry and pulsed-field and standard agarose gel electrophoresis. The experiments were performed using deproteinized calf thymus DNA in buffered deaerated aqueous solutions. A dual dose response was observed: a tendency for increased helix stability at "low" doses (0-4 Gy) accompanied by a high tendency of the DNA molecules to interact, forming larger molecules, followed by a gradual increase of degradation and helix instability at higher doses. The results reported here for the low-dose region are consistent with the hypothesis of inter- and intramolecular interactions of covalent character (crosslinking) in irradiated DNA molecules.

Diversity of peripheral blood mononuclear cells as revealed by a novel multiple microgel "comet assay".

Multiple microgel comet assay (MMCA) is a metho-dological adaptation of the single-cell gel electrophoresis assay in which we have introduced the use of standard agarose plug molds in an attempt to improve and expand the applications of the assay. We focused on the study of the heterogeneity of peripheral blood mononuclear cells (PBMC) at the level of the basal single-strand breakage and the DNA damage induction caused by ionizing radiation. Differences among subpopulations were also investigated at the level of chromatin organization and methylation after NotI digestion of microgel-embedded cells. In parallel experiments, the NotI-digested nucleoids were also analyzed with the use of pulsed-field gel electrophoresis (PFGE) and the DNA migration patterns were compared with the corresponding patterns from the MMCA. Significant heterogeneity in the distribution of the oxidative DNA damage, as well as intracellular variations in the NotI digestion patterns were observed in the cell population of PBMC. The combined use of both the comet assay and PFGE provides a useful model for analysis of variation in DNA damage in individual cells as well as information on size of DNA fragments.

Structure and morphogenesis of the eggshell and micropylar apparatus in the olive fly, Dacus oleae (Diptera: Tephritidae).

The egg of the olive fly, Dacus oleae (Diptera, Tephritidae), is laid inside olives and the larva eventually destroys the fruit. The oocyte is surrounded by several distinct layers which are produced during choriogenesis. The chorion covering the main body of the egg outside of the vitelline membrane includes a "wax" layer, an innermost chorionic layer, an endochorion consisting of inner and outer layers separated by pillars and cavities similar to their counterparts in Drosophila melanogaster, as well as inner and outer exochorionic layers. The anterior pole is shaped like an inverted cup, which is chiefly hollow around its base and has very large openings communicating with the environment. Holes through the surface of the endochorion result from deposition of endochorionic substance around follicular cell microvilli. An opening at the apex of the cup provides an entrance for sperm entering the micropylar canal, which traverses the endochorion and continues into a "pocket" in a thickened vitelline protrusion. The micropylar canal is formed by deposition of endochorion and vitelline membrane around an elongated pair of follicular cell extensions. These extensions later degenerate and leave an empty canal about 5 microns in diameter and the narrower pocket about 1 micron in diameter. Respiration is thought to be facilitated by openings at the base of the anterior pole as well as by openings through the "plastron" around the main body of the shell.

The egg-shell of Drosophila melanogaster III. Covalent crosslinking of the chorion proteins involves endogenous hydrogen peroxide.

Utilizing two cytochemical methods, namely, diaminobenzidine for the assay of peroxidases and cerium(III) chloride for the localization of hydrogen peroxide it was found that the enzyme exists in two out of the five egg-shell layers: the innermost choronic layer and the endochorion. In addition, hydrogen peroxide which acts as a substrate for the enzyme in vitro enabling the formation of covalent bonding between the egg-shell proteins, was found to be produced at the follicle cell plasma membrane during the last stage of oogenesis. It is concluded that hydrogen peroxide is an endogenous, programmed product of the follicle cells, responsible for the action of peroxidase in order to oxidize the tyrosyl residues producing di-tyrosine and tri-tyrosine bonds between the chorion polypeptides.

The eggshell of the cherry fly Rhagoletis cerasi.

One of the major pests in Greek cherry orchards is the cherry fly Rhagoletis cerasi (Diptera: Tephritidae). In order to complete our comparative work on the chorion assembly of other representatives of the fruit flies (e.g. Ceratitis capitata and Dacus oleae) we studied eggshell morphogenesis in the cherry fly. The oocyte is surrounded by several distinct layers which are produced during choriogenesis. The eggshell consists of the vitelline membrane, a fibrous layer of possible water-proofing function, an innermost chorionic layer, endochorionic and exochorionic layers. The endochorion shows a branched configuration with irregular cavities, and the exochorion consists of inner and outer layers for better embryo protection. At the anterior region of the follicle, the hexagonal borders of the follicle cells are created by endochorionic material, covered by both inner and outer exochorion. This area resembles the D. melanogaster chorionic appendages and therefore can serve for plastron respiration. The structural results support the phylogenetic relationships among the tephritids (Rhagoletis is closer to Ceratitis than Dacus). The presence of peroxidase in the endochorion, detected by diaminobenzidine, is consistent with the eggshell hardening at the end of choriogenesis, following the same pattern with the other fruit flies studied so far. Two major chorionic proteins are found both in R. cerasi and in C. capitata and therefore general conclusions can be drawn from this study, concerning the pattern of choriogenesis, which all dipteran insects follow, in order to create a resistant and functional eggshell, and the high conservation of the proteinaceous components of the chorion among species in the order.

The eggshell of the almond wasp Eurytoma amygdali (Hymenoptera, Eurytomidae) - 1. Morphogenesis and fine structure of the eggshell layers.

The almond wasp Eurytoma amvgdali (Hymenoptera: Eurytomidae) feeds and oviposits exclusively in almonds and therefore is characterized as an insect of economic importance. Its meroistic polytrophic ovaries include follicles with a tri-partite configuration. The mature follicles exhibit two filaments occupying the two poles of the egg. One is the micropylar filament while the other might serve for respiration since it is likely that its flattened end layers remain outside the almond fruit. The eggshell is formed by aposition and the follicle cells, which surround the follicle until the end of oogenesis, may be responsible for protein synthesis and secretion which finally lead to the assembly of the eggshell. The eggshell comprises the thin vitelline membrane, possibly a 'wax' layer of waterproofing function, a transluscent layer which appears amorphous even at the end of choriogenesis, a granular layer, including large and small electron-dense granules, and finally a columnar layer very similar to layers found in other insect species of the same or different orders. Peroxidase is histochemicalLY found for the first time in an eggshell of the Hymenoptera order: the tranluscent layer in particular is positively stained (electron-dense). Two possible roles of this peroxidatic activity are discussed, first, in comparison to other fruit-infesting insects, we assume that elastic chorion is produced through the function of peroxidase induced bonds (resilin-type bonds), very important for avoiding premature breaking, while being oviposited through a narrow ovipositor. Second, referring to other studies, this layer can play a bactericidal role for additional embryonic protection.

The eggshell of the almond wasp Eurytoma amygdali (Hymenoptera, Eurytomidae) - 2. The micropylar appendage.

Micropylar apparatuses in insects are specialized regions of the eggshell through which sperm enters the oocyte. This work is an ultrastructural study and deals with the structure and morphogenesis of the micropylar appendage in the hymenopteran Eurytoma amygdali. The micropylar appendage is a 130 microm long cylindrical protrusion located at the posterior pole of the egg, unlike other insects i.e. Diptera. in which the micropylar apparatus is located at the anterior pole. In mature eggs there is a 0.4 microm wide pore (micropyle) at the tip of the appendage leading to a 6 microm wide micropylar canal. The canal contains an electron-lucent substance, it travels along the whole appendage and finally reaches the vitelline membrane of the oocyte. The vitelline membrane is covered by a wax layer and an electron-lucent layer, whereas the chorion surrounding the canal consists of a granular layer (fine and rough) and a columnar layer. The morphogenesis of the appendage starts in immature follicles: four central cells located at the posterior tip of the oocyte near the vitelline membrane, differing morphologically from the adjacent follicle cells. These central cells degenerate during early chorionic stages, thus assisting in the formation of the micropylar canal. The adjacent, peripherally located cells secrete the electron-lucent substance which fills the canal and at the same time, the fine granular layer is formed starting from the base towards the tip of the appendage. The secretion persists at late chorionic stages and results in the formation of the chorion around the micropylar canal. The extremely long (compared to other insects) micropylar appendage seems to facilitate the egg passage through the very thin and long ovipositor. The structure and morphogenesis of this appendage differs significantly from the micropylar apparatuses studied so far in other insects i.e. Diptera, and may reflect adaptational and evolutionary relationships.

The eggshell of Drosophila melanogaster: IX. Synthesis and morphogenesis of the innermost chorionic layer.

Synthesis and morphogenesis of the innermost chorionic layer (ICL) was investigated by conventional EM methods, freeze-fracturing, tissue culture in Robb's medium, and EM autoradiography. Both autoradiography and fine structure results have shown that ICL-components are secreted prior to other chorion proteins. Their secretion starts on stage 12a but the first layer of ICL molecules is visible at stage 12b. Its thickness is gradually increased during the next stages, taking first, a bilaminar form along with the inner endochorion. Later, at the end of choriogenesis, ICL is detached from the endochorion and takes its final thickness and configuration, consisting of a 3-dimensional crystal, about 40 nm thick. The isolated ICL in conditions of air water interface is a monolayer crystal 10 nm thick. Studies on chorion mutants showed that the amount of protein secreted by the follicle cells is independent to the process of crystallisation. These data show how a proteinaceous extracellular substance is gradually assembled to form a 3-D crystal and how it can be organised to perform functions such as the physiological resistance of the insect eggs against water loss or water uptake, whenever they are laid on substrates with extreme environmental conditions. These functions are performed by ICL in conjunction with the underlying wax layer.

The egg-shell of Drosophila melanogaster. VI, Structural analysis of the wax layer in laid eggs.

Utilizing freeze-fracturing conventional electron microscopy and scanning electron microscopy methods, a wax layer was identified, sealing the oocyte of Drosophila melanogaster. In mature egg-shells wax forms a hydrophobic layer surrounding the oocyte and lying between, and in very close contact with the vitelline membrane (interiorly) and the crystalline intermediate chorionic layer (exteriorly). In cross-fractured views it is less than 50 A thick whereas in longitudinal fracturing it reveals smooth fracture faces of a multilayered material in the form of hydrophobic areas or plaques (0.5-1 microns in diameter) which are partially overlapping and highly compressed between the vitelline membrane and the innermost chorionic layer. The evidence for this layer being a wax are the facts that a) it is not preserved in conventional fat-extracting electron microscopy methods, b) it directs laterally the fracture planes during freeze-fracturing and reveals smooth fracture faces. Analysis of the structural features of wax in mature egg-shell in various species of Drosophilidae have shown that the wax layer exhibits indistinguishable (among the species) hydrophobic plaques, which have the same size and thickness with Drosophila melanogaster. These data provide structural evidence explaining the physiological resistance of the insect eggs studied, against water loss or water uptake, whenever they are laid on substrates with extreme environmental conditions. In addition, the data demonstrate how an extracellular substance can be organized to perform that function.

The eggshell of Drosophila melanogaster. VIII. Morphogenesis of the wax layer during oogenesis.

Utilizing freeze-fracturing and conventional electron microscopy methods, we have studied the details of morphogenesis and construction of the wax layer envelope from Oregon R and mutants of Drosophila melanogaster eggs during oogenesis. The wax layer is synthesized and secreted by the follicular cells in the form of lipid vesicles during stage 10b. During secretion (stages 10b, 11 and 12) the lipid vesicles are accumulated on the vitelline membrane surface and become flat. At the late stages of choriogenesis (stages 13, 14) the lipid vesicles are compressed tightly between the vitelline membrane and the other already constructed eggshell layers, so the wax layer becomes very thin and is hardly seen in cross-fractured views.

Quantitative and comparative ultrastructure of the vertebrate cornea. I. Urodele Amphibia.

The cornea of the urodele amphibian Triturus c. cristatus was studied ultrastructurally in order to provide the basis for a comparison among corneas throughout the vertebrate phylum. The cornea of this salamander consists of relatively thick epithelium and basement membrane and thin Descemet's membrane, unlike the mammalian corneas. The outermost epithelial cells contain Ruthenium Red stainable extracellular filaments and intracellular vesicles which are thought to play a role in the process of lubricating the corneal surface. Occluding junctions have been observed in the apical region of the superficial epithelial cells and are considered as barriers to the intercellular passage of material. A thin substantia propria (stroma) consists of about 40 collagenous highly organized lamellae. The thicknesses of the basement membrane, Descemet's membrane and the epithelium are believed to represent the primitive situation in the process of corneal evolution.

Quantitative changes and ultrastructural alterations of the cornea in response to ultraviolet light. II. Effects of amphibia; elucidation of desmosomal structure and basement membrane synthesis.

The effects of far ultraviolet light irradiation upon an amphibian cornea were studied to compare the effects observed both quantitatively and ultrastructurally with data obtained after UV irradiation of mammalian corneas. The ultimate goal of this series of investigations is the elucidation of the alterations and the regeneration mechanisms, which might reflect existing morphological diversities among the species, observed in vertebrate corneas following exposure to UV light. It was found that while the epithelial cells undergo oedema after low dose exposures and are gradually damaged after high doses of UV light, 2-4 days leter a new epithelium has been formed. Intercellular permeability is increased by low dose exposure as was detected by the penetration of Ruthenium Red into the intercellular clefts. Under these conditions desmosomal structure revealed a 21-laminar configuration. The basement membrane of the amphibian, unlike that of the mammal, does not dissolve away upon exposure but shows localized disruptions which are thought to accommodate the passage of leucocytes from stroma to epithelium. That a new basement membrane is subsequently formed is evident by the existence of extracellular and intracellular secretion granules. In comparison to irradiated rabbit corneas, this stroma remains remarkably at the same thickness following a high dose exposure although a noticeable disorganization of collagen arrangement is apparent. Finally, as in the case of the rabbit corneas, a secondary degeneration of endothelium was observed 4 days after a moderate dose exposure.

Fine structure of the chorion of Manduca sexta and Sesamía nonagrioides as revealed by scanning electron microscopy and freeze-fracturing.

The fine structure of Manduca sexta and Sesamia nonagrioides chorion was investigated by scanning electron microscopy and freeze-fracturing. In both species the mature chorion exhibits a complex ultrastructure on its outer surface, with a large number of aeropyles forming polygonal arrays. The micropyle is surrounded by a rosette of approximately 80 follicular cell imprints. Scanning electron microscopy of vertically ripped sections reveals that both chorions consist of two main layers: a trabecular layer closest to the oocyte and a lamellar layer. The technique of freeze-fracturing, utilizing single-sided and rotary shadowing, clearly shows that fibrils, approximately 3-4 nm in diameter, constitute chorionic lamellae in both species. The fibrils appear to have a 'beaded' structure, with a 2-3 nm axial periodicity. Freeze-fracturing also provides a direct visualization of the helicoidal arrangement of these fibrils for the formation of chorion supramolecular architecture.

Three-dimensional reconstruction of innermost chorion layer of Drosophila grimshawi and Drosophila melanogaster eggshell mutant fs(1)384.

A low-resolution three-dimensional structure of the crystalline innermost chorionic layer (ICL) of the Hawaiian species Drosophila grimshawi and the Drosophila melanogaster eggshell mutant fs(1)384 has been calculated from electron microscope images of tilted negatively stained specimens. The isolated ICL of Drosophila grimshawi is a three-layer structure, about 36 nm thick, whereas the ICL of Drosophila melanogaster eggshell mutant fs(1)384 is a single layer, about 12 nm thick. Each unit in both crystalline structures includes octamers made up of four heterodimers. Crosslinks between the structural elements, both within and between unit cells form an interconnecting network, apparently important in maintaining the integrity of the layer. A model which may account for the ICL self-assembly formation in vivo and the ICL observed lattice polymorphism is proposed, combining data from the three-dimensional reconstruction work and secondary structure features of the ICL component proteins s36 and s38.

Short-term memory in mice is affected by mobile phone radiation.

The effects of mobile phone electromagnetic fields (EMFs) were studied on a non-spatial memory task (Object Recognition Task - ORT) that requires entorhinal cortex function. The task was applied to three groups of mice Mus musculus C57BL/6 (exposed, sham-exposed and control) combined with 3 different radiation exposure protocols. In the first protocol designated "acute exposure", mice 45 days old (PND45 - postnatal day 45) were exposed to mobile phone (MP) radiation (SAR value 0.22W/kg) during the habituation, the training and the test sessions of the ORT, but not during the 10min inter-trial interval (ITI) where consolidation of stored object information takes place. On the second protocol designated "chronic exposure-I", the same mice were exposed for 17 days for 90min/per day starting at PND55 to the same MP radiation. ORT recognition memory was performed at PND72 with radiation present only during the ITI phase. In the third protocol designated "chronic exposure-II", mice continued to be exposed daily under the same conditions up to PND86 having received radiation for 31 days. One day later the ORT test was performed without irradiation present in any of the sessions. The ORT-derived discrimination indices in all three exposure protocols revealed a major effect on the "chronic exposure-I" suggesting a possible severe interaction of EMF with the consolidation phase of recognition memory processes. This may imply that the primary EMF target may be the information transfer pathway connecting the entorhinal-parahippocampal regions which participate in the ORT memory task.