Epipolarization Microscopic Immunogold Assay: a Combination of Immunogold Silver Staining, Enzyme-Linked-Immunosorbent Assay and Epipolarization Microscopy

We describe a new immunoassay which combines an immunosorbent assay, Immunogold silver staining and epipolarization microscopy. Our new assay procedure features multiple samples on a single microscope slide, and high sensitivity of epipolarization microscope for detection of silver-enhanced colloidal gold as a final immunoassay product. We call the new immunoassay “on slide immunogold assay” (OSIGA). This new method uses biotinylated antibody and streptavidin-gold reaction with silver enhancement technique. With OSIGA it is possible to investigate 30 samples on a single microscopic slide. Our preliminary studies used 10-20 μ1 samples and detected nanogram quantities of a standardized protein solution. Unlike enzyme linked immunosorbent assay (ELISA), which has a limited time for reading the final color products, the OSIGA specimens can be dried or resin mounted for longer storage and future reference.     

Nuclear matrix involvement in sperm head structural organization

Summary— In the sperm nuclei the DNA is packaged into a highly condensed form and is not organized into nucleosome and solenoid but is bound and stabilized mainly by the protamines that arrange the DNA in an almost crystalline state. As demonstrated for somatic cells, the sperm DNA has been reported to be organized in loop domains attached to the nuclear matrix structures. However, the possible role of the sperm head matrix in maintaining the loop organization in absence of a typical nucleosomal structures has not been fully elucidated. By using in situ nick translation at confocal and electron microscope level, we analyzed the organization of the DNAprotamine complex and its association with the sperm nuclear matrix. The data obtained indicate that the chromatin organization in sperm nuclei is maintained during the sperm condensation by means of interactions with the nuclear matrix at fixed sites. The fine stucture of sperm nucleus and of sperm nuclear matrix, investigated on sections and replicas of freeze-fractured specimens, suggests that the lamellar array, observed by freeze-fracturing in the sperm nuclei, could depend on the inner matrix which presents a regular organization of globular structures possibly involved in the maintenance of chromatin domains in highly condensed sperm nuclei also.     

Fluorescence lifetime analysis of DNA intercalated ethidium bromide and quenching by free dye

The fluorescence characteristics of ethidium bromide (Eb) complexed to calf thymus DNA have been examined using fluorescence lifetime analysis for a range of DNA (effective nucleotide concentration) to Eb molar ratios. Control of both temperature and ion concentration is necessary for reproducible analyses. Eb complexed to double stranded DNA has a maximum fluorescence lifetime of 23 ns and is easily distinguishable from a fluorescence lifetime value of 1.67 ns corresponding to unbound Eb. In a solution of calf thymus DNA containing excess Eb a binding equilibrium is reached, and this corresponds to one Eb molecule for every five nucleotides. With increasing amounts of unbound Eb, the fluorescence lifetime of the DNA-Eb complex decreases with a concomitant drop in the steady state fluorescence intensity, without a change in the amount of Eb bound to DNA. It is concluded that unbound Eb, acting via a quenching mechanism, shortens the fluorescence lifetime of bound Eb and consequently decreases the overall fluorescence intensity. This means that a different approach is necessary: time-resolved fluorescence spectroscopy directly distinguishes between a decrease in fluorescence intensity due to quenching by an excess of unbound Eb from that due to a decrease in Eb binding to double-stranded DNA. These studies suggest that techniques which measure total steady state fluorescence intensity of bound Eb in order to infer relative amounts of double-stranded DNA must be interpreted with caution. For such assays to be valid it is essential that no unbound Eb be present; otherwise a variable correction factor is required to account for unbound Eb.     

Effects of cytochalasin D on the distribution of actin and ACTH-induced steroidogenesis in cultured embryonic adrenal gland cells from the Pekin duck (Anas platyrhynchos)

Cultured steroidogenic cells derived from the adrenal glands of duck embryos were used to study changes in the distribution of actin associated with the corticotropic responsiveness. Actin-containing components were identified by rhodamine-phalloidin staining. The actin in most of the unstimulated cells occurred as stress fibers that either ran parallel throughout the cell or were present as domains of parallel fibers at angles to one another. When incubated in Krebs-Henseleit buffer containing 1–24 ACTH, the cells released approximately equal amounts of corticosterone and aldosterone. Incubation of the cells in buffer containing cytochalasin D caused the cells to lose their stress fibers, and the actin became distributed at the periphery in what appeared to be fragments of stress fibers and clumps of fibrous material in the central cytoplasm. Although cytochalasin D did not affect the basal output of corticosterone and aldosterone, the 1–24 ACTH-induced rates of both hormones were suppressed significantly. After the cells had been washed in unadulterated buffer, the normal distribution of actin stress fibers was restored and the cells responded normally when incubated in buffer containing 1–24 ACTH. These results suggest that the actin components of the cytoskeleton are important determinants of corticotropin-induced steroidogenic responsiveness.     

Fine structure and lectin histochemistry of the apical surface of the free neuromast of Lampetra japonica

The surface coat, ciliary process, and microvilli of the lamprey neuromast were examined with electron microscopy after tannic acid prefixation and lectin histochemistry. The neuromast was found to exist in the form of a dermal mound with a furrow in the middle. On the bottom of the furrow, the hair cell was characterized by a kinocilium and 15–20 stereocilia, arranged along the longitudinal axis of the furrow. Spanning structures were demonstrated between the kinocilium and stereocilia as well as between stereocilia. The surface coat, enhanced by tannic acid prefixation, was particularly rich over the surface of the supporting cell; by contrast, it was thin over the hair cell. Some lectins (PNA, GS-I, SBA, WGA) showed affinity to the surface coat of the supporting cell as well as the hair cell, and the others (RCA-I, MPA, ConA) showed affinity only to the supporting cell. These differences in the structure and affinities of the surface coat suggest an extracellular milieu highly specialized for the hair cell in this particular form of the mechanoreceptor.     

Astrocytes alter their polarity in organotypic slice cultures of rat visual cortex

The ultrastructure of astrocytes in an organotypic slice culture of the rat visual cortex was investigated using ultrathin sections and freeze-fracture replicas. After a culture period of 9–15 days, a glial scaffold formed that separated the bulk of the slice neuropil from the medium and the underlying plasma clot. However, the glial cells and processes did not build a dense barrier but allowed the outgrowth of neurites. A basal lamina covering the medium-oriented surface of the astrocytes was not found. In freeze-fracture replicas, orthogonal arrays of particles (OAP) were characteristic components of astrocytic membranes. The OAP density in membranes bordering the medium was 35±13 OAP/m², corresponding to 2.5% of this membrane area; the OAP density in membranes within the slice neuropil was 22±12 OAP/², corresponding to 1.4% of this membrane area. Although the difference was significant, it was greatly reduced when comparing OAP densities in endfoot and non-endfoot membranes in vivo. Another mode of polarity was recognized in astrocytes of the organotypic slice culture. In membranes of astrocytes bordering upon the medium, the density of non-OAP intramembranous particles (IMP) was clearly higher (1130±136 IMP/ m²) than in membranes of astrocytes in the center of the slice (700±172 IMP/m²). This pronounced IMP-related polarity was observed neither in vivo nor in cultured astrocytes. The present study suggests, together with data from the literature, that the distribution of astrocytic OAP across the cell surface is influenced by the existence of a basal lamina and neuronal activity, and that astrocytes possess a more remarkable plasticity of membrane structure than previously suspected.     

A comparative study of the glomerular peripolar cell and the renin-secreting cell in twelve mammalian species

The peripolar cell is a glomerular epithelial cell situated within Bowman's capsule at its vascular pole. It is believed to be a secretory cell which forms part of the juxtaglomerular apparatus. Scanning electron microscopy was used to perform a comparative study of the morphology and number of peripolar cells in twelve mammalian species. The number of renin-secreting cells in kidney sections stained by renin antibodies and immunocytochemistry was counted. There was a marked inter-species variation in the number, size and appearance of peripolar cells. They were largest and most abundant in sheep and goat and fewest in dog, cow and human. There was no correlation between the numbers of peripolar cells and renin-secreting cells. This does not support the view that the peripolar cell is part of the juxtaglomerular apparatus.     

Neosteinernema longicurvicauda n. gen., n. sp. (Rhabditida: Steinernematidae), a Parasite of the Termite Reticuldermes flavipes (Koller)

A nematode isolated from the termite Reticulitermes flavipes (Koller) was identified and described as a new genus and species, Neosteinernema longicurvicauda. Primary distinguishing characters, by contrast to members of the genus Steinernema, were females having prominent phasmids, a curved tail longer than the body width at the anus, a spiral shape in juvenile-bearing females, and juveniles becoming infective-stage juveniles before emerging from the female; males having prominent phasmids, a digitate tail tip, a characteristic shape of the spicules (foot-shaped with a hump on the dorsal side), and 13-14 pairs of genital papillae, with eight pairs preanal; and infective juveniles having prominent phasmids and a filiform curved tail as long as the esophagus. Adult nematodes are found outside the termite cadaver. Diagnosis of the family Steinernematidae was emended to accommodate the new species.     

Descriptions of Two New Species of Chronogaster Cobb, 1913 from India

Two new species of Chronogaster in India were described and illustrated, based on light and scanning electron microscopy. Chronogaster neotypica n. sp. collected from a sewage slurry was characterized by a medium-sized body, a ventral tail mucro without additional spines, absence of longitudinal incisures in lateral fields, and by the presence of crystalloids in the body. Diagnostic for C. spinicauda n. sp. collected from soil around roots of mango were a medium-sized body, a tail mucro with 10 spines, and absence of lateral lines and crystalloids. Males were not found.