The inner ear morphology and hearing abilities of the Paddlefish (Polyodon spathula) and the Lake Sturgeon (Acipenser fulvescens)

Concern regarding the spread of silver carp (Hypopthalmichthys molitrix) and bighead carp (Aristichthysc nobilis) through the Illinois River has prompted the development of an Acoustic Fish Deterrent (AFD) system. The application of this technology has resulted in a need to understand the auditory physiology of fish other than the target species, in order to minimise the effect of the AFD barrier on the ecology of indigenous fish populations. To this end, both the structures involved in sound reception and the hearing abilities of the paddlefish (Polyodon spathula) and the lake sturgeon (Acipenser fulvescens) are studied here using a combination of morphological and physiological approaches, revealing that both fish are responsive to sounds ranging in frequency from 100 to 500 Hz. The lowest hearing thresholds from both species were acquired from frequencies in a bandwidth of between 200 and 300 Hz, with higher thresholds at 100 and 500 Hz. The rationale for studying hearing in P. spathula and A. fulvescens in particular, is the value placed on them by both the commercial caviar producing industry and by the recreational fisheries sector. The hearing abilities of twelve P. spathula and twelve A. fulvescens were tested in sound fields dominated by either sound pressure or particle motion, with the results showing that acipenseriform fish are responsive to the motion of water particles in a sound field, rather than the sound pressure component. In this study, we measure the intensity of the sound field required to evoke threshold responses using a pressure sensitive hydrophone, as pressure dominated sound fields are the most audible acoustic condition for specialists like H. molitrix and A. nobilis (the target species). The results of the auditory examination clearly show that P. spathula and A. fulvescens are not sensitive to sound pressure, and will therefore have a significantly higher deterrent threshold than H. molitrix and A. nobilis in a pressure dominated sound field.     

匙吻鲟的细胞遗传学分析

采用体内注射PHA和秋水仙素,肾细胞短期培养,常规空气干燥法制备匙吻鲟(Polyodon spathula)的染色体标本.对其肾细胞染色体数目统计分析表明,匙吻鲟染色体组南120条染色体所组成.以测得的核型参数和按Levan,et al.提出的染色体划分标准得出:具有22对中部着丝粒染色体(m),16对亚中部着丝粒染色体(sm),22对亚端部着丝粒染色体(st)和端部着丝粒染色体(t);染色体臂数(NF)为196,核型公式为44 m + 32am + 44st,t.再采用流式细胞仪分析系统测定匙吻鲟体细胞的DNA含量,与鸡血细胞标准对照相比为2.69±0.19,以鸡红细胞DNA含量2.3 pg/N测算.则匙吻鲟体细胞DNA含量为6.18 pg/N.根据所得到的结果并结合已发表的鲟鱼类DNA含量和染色体资料,判定匙吻鲟为四倍体物种.鲟形目伍类全部为多倍体起源的鱼类,在细胞水平的进化比较特殊,以染色体加倍的方式进行.染色体加倍及分化,可能是造成鲟形目鱼类种类较多及多倍体类型(4n、8n、12n等)丰富的主要原因.     

Effect of some negative stains on the calcium transport of sarcoplasmic reticulum

Summary Effect of some negative stains used in electron microscopy on calcium-transporting membranes of the fragmented sarcoplasmic reticulum (FSR) and interaction between stains and calcium have been studied.Calcium transport as well as ATP-ase activity of FSR are affected by stains investigated at concentrations lower than those used in electron microscopy. Degree of alteration varies from stain to stain. Phosphotungstic acid is the most effective inhibitor.Calcium loaded vesicles are depleted during resuspension in various staining solutions, in concentration routinely used for electron microscopy, according to the calcium affinity of the stain. Also in this respect phosphotungstic acid is the most effective.     

Clonal Aging In Two Species of Spathidium (Ciliophora: Gymnostomatida)*

Thirty newly excysted Spathidium spathula (Müller) and 60 newly excysted Spathidium muscicola (Kahl) were selected as progenitors of clonal daily reisolation lines that omitted both conjugation and encystment. Daily division rates were determined for each clone either until it died or until the end of the 170 days of reisolation. Both species had reduced fission rates as they accumulated fissions in the absence of macronuclear reorganization. Spathidium spathula had a significant reduction of daily fission rate after 100-120 fissions and S. muscicola after 20-30 fissions. Older clones of both species contained a noticeable proportion of abnormal organisms. A significant increase (10.5%) in daily fission rate occurred in aged sublines of S. spathula following conjugation (selfing) and its concomitant nuclear reorganization. Spathidium muscicota did not conjugate, but recently excysted sublines, compared to aged lines, had an increased daily fission rate.     

HISTOCHEMICAL LOCALIZATION AT THE ELECTRON MICROSCOPE LEVEL

Albersheim, Peter, and Ursula Killias. (Harvard U., Cambridge, Mass.) Histochemical localization at the electron microsco pe level. Amer. Jour. Bot. 50(7): 732–745. Illus. 1963.—This paper discusses the use of chemical reactions for specifically locating cellular constituents with the electron microscope. Presented in detail are results obtained by using alkaline hydroxylamine and ferric chloride as an electron stain for pectin. This treatment also results in the staining of certain cytoplasmic and nucleolar particles of unknown nature. Also described is the use of bismuth as an electron stain for nucleic acids. A series of micrographs is presented depicting bismuth stained onion root tip cells in various stages of mitosis.     

The parasitic coelenterate, Polypodium hydriforme USSOV, from the eggs of the American acipenseriform Polyodon spathula.

No significant differences in macro- and micromorphology were found between the parasitic stolon and free-living polyps of Polypodium sp. obtained from infected eggs of the North American acipenseriform fish Polyodon spathula and corresponding developmental stages of Polypodium hydriforme Ussov, parasitic in the Volga sterlet (Acipenser ruthenus). Therefore, both the American and the European forms of Polypodium belong to the species P. hydriforme Ussov.     

A beaked basal ornithurine bird (Aves, Ornithurae) from the Lower Cretaceous of China

We report here one of the earliest known beaked ornithurine birds from the Lower Cretaceous deposits in Liaoning, northeast China. The new basal ornithurine, Archaeorhynchus spathula gen. et sp. nov., has a rhynchokinetic skull with toothless jaws. It also contains over three dozen preserved gizzard stones, suggesting an herbivorous diet. The distal end of the tibiotarsus is unfused, enabling recognition of the astragalus with a broad ascending process, generally similar to that of Archaeopteryx . The new discovery sheds new light on our understanding of the early radiation and diet diversification of early birds in the Lower Cretaceous.     

Application of olefin metathesis in the synthesis of steroids

Over the past decade, ruthenium-mediated metathesis transformations, including cross-metathesis, ring-closing metathesis, enyne metathesis, ring-opening metathesis polymerization, and also tandem processes, belong to the most intensively studied reactions. Many applications of olefin metathesis in the synthesis of natural products have been recently described. Also in the field of steroid chemistry new methods of total synthesis and hemisynthesis based on metathesis reactions have been elaborated. Various biologically active compounds, e.g. vitamin D and hormone analogues, steroid dimers and macrocycles, etc. have been prepared using a variety of olefin-metathesis protocols.     

Native gel activity stain and preparative electrophoretic method for the detection and purification of pyridine nucleotide-linked dehydrogenases

An activity stain for the detection of pyridine nucleotide-linked dehydrogenases in polyacrylamide gels is described. Following incubation of the gel with substrate and cofactor, bands are visualized under ultraviolet light, where reduced cofactors fluoresce and oxidized cofactors appear black. The methods described are useful for any NAD- or NADP-linked dehydrogenase; the enzymes can be assayed in either the oxidative or the reductive direction. Also described is a preparative polyacrylamide gel system using the activity stain, which can be used as a general purification method for dehydrogenases. The preparative gels are crosslinked with bisacrylylcystamine. These crosslinks can be broken by the addition of thiols after the bands of interest have been located and excised. The protein of interest is then separated from the solubilized acrylamide by adsorption to a suitable resin.     

Histology and ultrastructure of the coenenchyme of the octocoral Swiftia exserta,a model organism for innate immunity/graft rejection

The octocoral Swiftia exserta has been utilized extensively in our laboratory to study innate immune reactions in Cnidaria such as wound healing, auto- and allo-graft reactions, and for some classical “foreign body” phagocytosis experiments. All of these reactions occur in the coenenchyme of the animal, the colonial tissue surrounding the axial skeleton in which the polyps are embedded, and do not rely on nematocysts or directly involve the polyps. In order to better understand some of the cellular reactions occurring in the coenenchyme, the present study employed several cytochemical methods (periodic acid–Schiff reaction, Mallory's aniline blue collagen stain, and Gomori's trichrome stain) and correlated the observed structures with electron microscopy (both scanning and transmission). Eight types of cells were apparent in the coenenchyme of S. exserta, exclusive of gastrodermal tissue: (i) epithelial ectoderm cells, (ii) oblong granular cells, (iii) granular amoebocytes, (iv) morula-like cells, (v) mesogleal cells, (vi) sclerocytes, (vii) axial epithelial cells, and (viii) cnidocytes with mostly atrichous isorhiza nematocysts. Several novel organizational features are now apparent from transmission electron micrographs: the ectoderm consists of a single layer of flat epithelial cells, the cell types of the mesoglea extend from beneath the thin ectoderm throughout the mesogleal cell cords, the organization of the solenia gastroderm consists of a single layer of cells, and two nematocyst types have been found. A new interpretation of the cellular architecture of S. exserta, and more broadly, octocoral biology is now possible.     

Automatic tumor-stroma separation in fluorescence TMAs enables the quantitative high-throughput analysis of multiple cancer biomarkers

The upcoming quantification and automation in biomarker based histological tumor evaluation will require computational methods capable of automatically identifying tumor areas and differentiating them from the stroma. As no single generally applicable tumor biomarker is available, pathology routinely uses morphological criteria as a spatial reference system. We here present and evaluate a method capable of performing the classification in immunofluorescence histological slides solely using a DAPI background stain. Due to the restriction to a single color channel this is inherently challenging. We formed cell graphs based on the topological distribution of the tissue cell nuclei and extracted the corresponding graph features. By using topological, morphological and intensity based features we could systematically quantify and compare the discrimination capability individual features contribute to the overall algorithm. We here show that when classifying fluorescence tissue slides in the DAPI channel, morphological and intensity based features clearly outpace topological ones which have been used exclusively in related previous approaches. We assembled the 15 best features to train a support vector machine based on Keratin stained tumor areas. On a test set of TMAs with 210 cores of triple negative breast cancers our classifier was able to distinguish between tumor and stroma tissue with a total overall accuracy of 88%. Our method yields first results on the discrimination capability of features groups which is essential for an automated tumor diagnostics. Also, it provides an objective spatial reference system for the multiplex analysis of biomarkers in fluorescence immunohistochemistry.     

Eosinophils and mast cell homogeneity of the guinea pig eyelid skin, conjunctiva, and ileum

Mast cell heterogeneity has been described on the basis of differential staining reactions, light microscopic morphology, anatomic location, degranulation after polyamines, biochemical contents, growth requirements, and reactions to lymphokines. We have demonstrated typical "connective-tissue mast cells" by using anatomic criteria, histological staining reactions, electron microscopy, and reaction to compound 48/80 in the guinea pig conjunctiva, eyelid skin, and ileum. A second, much larger population of cells in the ileal mucosa and the conjunctiva, and rarely in the eyelid skin stained reddish-blue with acid toluidine blue in tissue fixed in ethanol-acetate-lead subacetate (BLA) and with alkaline Giemsa in formaldehyde-fixed tissue, did not stain with ethanolic or acid toluidine blue in formaldehyde-fixed tissue or with alkaline Giemsa in BLA-fixed tissue, and did not degranulate after 48/80 treatment. These are features of the rat intestinal "mucosal mast cells"; however, ultrastructural and light microscopic studies with the orcein Giemsa stain demonstrated these cells in the guinea pig to be eosinophils. Tissue culture, biochemical, and immunological studies indicate the existence of a second type of mast cell (bone-marrow-derived mast cell), ultrastructurally almost indistinguishable from the connective tissue mast cell. Our studies demonstrate only one mast cell type in the guinea pig and support the contention that other forms of mast cells are immature forms or variants of the connective-tissue mast cell.     

The Degradation of Romanowsky-Type Blood Stains in Methanol

The oxidative demethylation of Romanowsky-type stains in methanol has been examined quantitatively with respect to its effect upon the staining of blood smears. Spectral changes in hound dye, observed through two color filters, have been measured for the nuclei and cytoplasm of segmented neutrophils and monocytes utilizing the LARC™ automated differential analyzer. Stain decomposition in methanol results in a large loss in staining intensity with little change in color. The loss in intensity has been correlated with the observed spectral changes in the degraded stain. High-performance liquid chromatographic analysis of degraded stain samples has shown the products of methanolic degradation to be different from those obtained in aqueous polychroming reactions. To maintain a stain of defined thiazine dye composition and thus defined staining properties, refrigeration is recommended.     

The degradation of Romanowsky-type blood stains in methanol.

The oxidative demethylation of Romanowsky-type stains in methanol has been examined quantitatively with respect to its effect upon the staining of blood smears. Spectral changes in bound dye, observed through two color filters, have been measured for the nuclei and cytoplasm of segmented neutrophils and monocytes utilizing the LARC automated differential analyzer. Stain decomposition in methanol results in a large loss in staining intensity with little change in color. The loss in intensity has been correlated with the observed spectral changes in the degraded stain. High-performance liquid chromatographic analysis of degraded stain samples has shown the products of methanolic degradation to be different from those obtained in aqueous polychroming reactions. To maintain a stain of defined thiazine dye composition and thus defined staining properties, refrigeration is recommended.     

Histochemical observation of the halo on the epidermal cell wall of barley leaves attacked by Erysiphe graminis hordei

Summary Histochemical characters of the halo on the epidermal cell wall of barley leaves attacked byErysiphe graminis hordei were investigated with light microscope. The area corresponding to the halo showed reduced intensity of stain reactions for cuticle, pectin and cellulose, especially at the penetration point. From the several histochemical reactions, it was concluded that reducing substances with aldehyde groups (such as hexose), pentoses and possibly uronic acids occur in the halo. According to these results, it can be concluded that the halo is caused by the degeneration of the epidermal cell wall by the fungal enzymes.Contributions from Laboratory of Plant Pathology, Kyoto Univ., No. 230.     

Silver staining of proteins on electroblotting membranes and intensification of silver staining of proteins separated by polyacrylamide gel electrophoresis

A fast and convenient method for silver staining of proteins on electroblotting membranes was developed based on Gallyas' histochemical intensifier and applied to human endothelial cell proteins separated by one- and two-dimensional electrophoresis and electroblotted to polyvinyl difluoride membranes. The method allowed detection of proteins on membranes with a sensitivity equal to the sensitivity of the most sensitive silver-staining protocols for electrophoresis gels. Also, the method was compatible with preceding immunostaining on the same membrane. Furthermore, an intensifying method for proteins in silver-stained SDS-PAGE gels was developed based on Gallyas' histochemical intensifier. This method was applied to proteins separated by one- and two-dimensional gel electrophoresis and visualized by one of several silver-staining methods. Maximal intensification was achieved for the less sensitive but fast acidic silver-staining protocols, but even for the very sensitive alkaline protocols a significant increase in signal to noise ratio was obtained. In particular, negatively stained or invisible proteins on the silver-stained gels were found to be visualized by the Gallyas stain. Proteins from silver-stained and Gallyas-stained gels were identified by mass spectrometry, and the intensification procedure was fully compatible with mass spectrometry.     

Fresh Tissue Diagnosis in the Operating Room

A polychromatic stain giving color reactions closely simulating hematoxylin and eosin, without overstaining and obscuring the finer detail of the cell, has been developed in this laboratory. It is as follows: To 4 parts of 1% aqueous azure A (azure I) which has been previously filtered, add very rapidly one part of filtered 0.5% aqueous Erie garnet B. The mixture is immediately filtered to prevent precipitation. Occasional refiltering may be necessary if the mixture has been standing a month or more. Float tissue slices from a freezing microtome directly into distilled water; pick them up with a round glass rod and immerse into the stain which has been poured into a small section dish. The staining time varies with different tissues but 10 to 15 seconds usually gives the most satisfactory results. Lift sections from the stain, still on the same glass rod, float thru two changes of distilled water, and then transfer to a clean glass slide. Place a large drop of 40% glucose on a clean cover slip and immediately invert over the section. The mounted specimen is then ready for microscopic examination. In examining the tissues under the microscope it is best to employ an intense artificial light (an ordinary 40 or 60 watt bulb is sufficient). After mounting, one-half to one minute should elapse before examining the section to allow it to clear.     

Heteroplasmy in the mtDNA control region of sturgeon (Acipenser, Huso and Scaphirhynchus)

Data from 1238 fishes from 19 sturgeon species and 1 paddlefish were used to analyze heteroplasmy in sturgeon. Lengths of central repeat units ranged from 74 to 83 bp among sturgeon species. No repeat sequence was found in the paddlefish, Polyodon spathula. A general feature of the repeat units was the presence of termination associated sequence (TAS) motifs. About 50% of 138 interspecific mutations observed among the D-loop sequences are located 10 bp down- and upstream from these TAS motifs. Interestingly, most homoplasmic species showed deletions upstream to the TAS motifs, whereas deletions downstream to the TAS motifs observed in two species do not seem to preclude heteroplasmy. Calculations of secondary structures and thermal stabilities of repeat units showed DeltaG values for all heteroplasmic species to be <-8 and for most homoplasmic species DeltaG value to be >-8. Most heteroplasmic fishes had two and/or three repeat units. No homoplasmic sturgeon with >2 repeat units were observed. Molecular phylogeny based on the entire cytochrome b showed that heteroplasmy probably resulted from a single evolutionary event. Our data demonstrate that heteroplasmy is present in most sturgeon species and suggest that the thermal stability of the secondary structure of the repeat unit in combination with mutations downstream of the TAS sequences influences heteroplasmy.