Microscopical methods promote the understanding of the integument biology of Rhinella ornata

In Rhinella ornata, the integument of different body regions was investigated using low-vacuum scanning electron and light microscopy through histochemical and immunohistochemical methods, and revealed the basic structure found in other anurans. Keratinocytes formed the keratinized squamous stratified epithelium, and flask cells occurred among the epidermal superficial layer. Just below the epidermis, the dermis was subdivided into a spongious dermis and a compact dermis. Mixed and granular glands were located in the spongious dermis, and myoepithelial cells surrounded their secretory portions. The Eberth-Katschenko (EK) layer occurred as basophilic areas between the spongious and compact dermis and throughout the spongious dermis. A series of alternating layers of bundles of collagenous fibers characterized the compact dermis, being firmly attached to a thin hypodermis. Regarding the morphological features, each integument region revealed distinct structural aspects. The dorsal integument was rougher than ventral, having conical tubercles and spines upon the verrucae. In the ventral and subgular regions, the slightly elevations are visualized, tubercles were absent but some spines occurred. The pelvic integument had a non-keratinized epidermis with an irregular profile and small poorly developed elevations. In addition, the EK layer was absent, and the presence of numerous blood vessels suggests its participation in water absorption and distribution. In the parotoid gland, the epidermis was similar to the dorsal integument; except that the reticular dermis was present, and contained three exocrine glandular types. Expression of cytokeratin was evident in the basal and intermediary layer, but absent in the cornified layer. Flask cells showed strong cytokeratin labeling. Consequently, the integument had the basic morphology, but exhibits regional characteristics, which may be related to the physiology of each surface.     

Structure of the sexually dimorphic gland of Cycloramphus fuliginosus (Amphibia, Anura, Cycloramphidae)

Cycloramphus fuliginosus males (Amphibia, Leptodactylidae) have discoid glandular elevations on the abdominal inferior body region which are considered a characteristic of genus. In this work, this sexually dimorphic gland of C. fuliginosus was investigated by low vacuum scanning electron microscopy, light microscopy, and transmission electron microscopy. In this cycloramphid, the covered integument exhibits the basic structure: the epidermis, a keratinized squamous stratified epithelium with flask cells, and the dermis. The dermis is subdivided into spongious and compact dermal layers. In the iliac gland region, an aggregate of small mucous glands and larger syncytial tubuloalveolar glands occur in the spongious dermis, and they do not go beyond the Eberth-Katschenko layer limit. The adenocytes of the mucous gland produce neutral glycoproteins, in contrast to the larger tubuloalveolar glands that elaborate a proteinaceous secretion. Myoepithelial cells surround the alveoli, and play an important role in the secretion extrusion. The final secretion, elaborated by the iliac gland, is a mixture of mucus and protein. Both secretions are important to the cycloramphid biology, and may act as pheromone and/or as chemical parental care.     

Morphological characterization of the anuran integument of the Proceratophrys and Odontophrynus genera (Amphibia, Anuran, Leptodactylidae)

The morphological characteristics of the leptodactylid integument of Proceratophrys and Odontophrynus genera were investigated by means of stereoscopic, low vacuum scanning electron and light microscopy. The integument surface of Proceratophrys boiei, Proceratophrys laticeps and Proceratophrys appendiculata exhibited several projections, while the integument of Odontophrynus americanus had rounded elevations with smooth profile. Light microscopic observations showed the basic integument morphology for all anurans, i.e., an epidermis and a dermis, which is subdivided into a spongious layer and a compact layer. The epidermis is formed by basal, intermediary and cornified layers. However, in Proceratophrys genus the cornified layer had an irregular outline, while in O. americanus the external surface was smooth. In the spongious dermis, mucous and venom exocrine glands were observed, but in O. americanus an exclusive glandular type with apocrine secretory pattern was identified. The integument morphology showed peculiar characteristics that may be helpful for genus distinction. Thus, morphological methods may be considered as an efficient means to characterize and to differentiate anuran genera.     

Dermal collagen organization in Bufo ictericus and in Rana catesbeiana integument (Anuran, Amphibian) under the evaluation of laser confocal microscopy

Collagen structural organization plays an important role in the mechanical property of the vertebrate integument. Bufo ictericus and Rana catesbeiana integument was investigated by light microscopy and laser confocal microscopy. Collagenous elements of the dermis were statistical analyzed. The integument is formed by the keratinized squamous stratified epidermis supported by the dermis that is subdivided into the spongious layer with a loose arrangement, and the compact layer formed by collagenous fibers arranged compactly in a criss-crossed manner. Thick collagenous columns have a perpendicular trajectory, and are formed by the assembling of alternating collagenous lamellae in both animals. Short intercolumns of collagenous fibrils connecting collagenous lamellae obliquely or transversally are observed in R. catesbeiana dorsal integument. The present study provides evidences that B. ictericus and R. catesbeiana integument has well-organized compact dermis, constituted by collagenous lamellae in a plywood manner. The integument organization is in contrast to the literature in some aspects. This dermal arrangement is important to the biomechanical property of both anuran integuments.     

Morphology of Bufo ictericus integument (Amphibia, Bufonidae)

Bufo ictericus integument was investigated by stereoscopic, low vacuum scanning electron microscopy, transmission electron microscopy and light microscopy. The studies revealed, that the dorsal integument surface is rougher than ventral. Three types of projections are visualized: larger rounded verrucae, smaller conical cornified tubercles, and conical short spines. Prominent verrucae are observed on the dorsal surface, being flatter on the ventral surface. The tubercles are visualized only on the dorsal surface. The verrucae are separated by grooves that may contribute spreading and retention of the glandular secretion upon the integument. The pattern of the epidermal grooves is also important for water distribution, protecting the animal against desiccation. The epidermis is composed of a stratified epithelium with intraepithelial blood vessels, where keratinocytes predominate, but flask cells, and Merkel cells also occur. In the spongious dermis, cutaneous glands are visualized. The compact dermis is a series of alternating layers of bundles of collagenous fibers, and between spongious and compact dermis there are basophilic areas that correspond to Eberth-Katschenko layer. The dorsal and the ventral surfaces of B. ictericus are morphologically distinct. The integument structure is related to the physiology of each surface and represents an adaptation to habitat, reflecting a lifestyle of the animal.     

Glycosaminoglycans and glycoconjugates in the adult anuran integument (Lithobates catesbeianus)

Glycosaminoglycans (GAGs) from the integument of Lithobates catesbeianus were biochemically characterized and histochemically localized. Moreover, carbohydrate distribution was investigated using conventional and lectin histochemistry at light microscopy. Hyaluronan (HA), dermatan sulfate (DS) and a heparanoid were found in the integument. Sulfated and carboxylated GAGs were visualized in the Eberth-Katschenko (EK) layer, in the mucous glands, in the hypodermis as well as in the mast cells. Furthermore, glucose and galactose were identified in the integument through thin layer chromatography (TLC) assays. N-Acetyl-β-glucosamine residues were identified in the mucous glandular cells, between the corneum and spinosum strata, in the subepidermal region, and in the EK layer. N-Acetyl-galactosamine residues were evident in the EK layer, corresponding to a residue of the dermatan sulfate chain, which may be related to the collagenous fiber arrangement. These glycoconjugates occurred as secretory glandular products and as dermal structural elements. Moreover, HA and DS are the predominant GAGs in the L. catesbeianus integument. Considering the importance of glycoconjugates, they play a significant role to the integrity of the skin, providing mechanical support for integument cells. In addition, they are important to the water regulation mechanisms, since L. catesbeianus is preferably aquatic.     

Effect of reaction systems and surfactant additives on the morphology evolution of hydroxyapatite nanorods obtained via a hydrothermal route

Well-dispersed hydroxyapatite (HA) nanorods with different morphologies were synthesized by a hydrothermal method in oleic acid, ethanol and water reaction system, and the surfactant assisted modifications effect was also comparatively studied. The structure and morphology of samples were characterized using X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy and transmission electron microscopy (TEM), respectively. The effect of reaction systems and surfactant additives on the morphology evolution of HA nanorods were discussed in detail. The results showed that the controlled experimental conditions in the systems, such as the content ratio of oleic acid/ethanol, pH value and the content ratio of Ca/P source had an significant effect on the morphology evolution of as-prepared HA nanorods. Further, the selected surfactant additives, such as cetyltriethylammnonium bromide (CTAB), sodium dodecyl sulfate (K12) also play an important role in the formation of the uniform morphology of HA nanorods. Some possible formation mechanisms of the HA nanorods in the present reaction systems is proposed.     

Biophysical Properties of Phenyl Succinic Acid Derivatised Hyaluronic Acid

Modification of hyaluronic acid (HA) with aryl succinic anhydrides results in new biomedical properties of HA as compared to non-modified HA, such as more efficient skin penetration, stronger binding to the skin, and the ability to blend with hydrophobic materials. In the present study, hyaluronic acid has been derivatised with the anhydride form of phenyl succinic acid (PheSA). The fluorescence of PheSA was efficiently quenched by the HA matrix. HA also acted as a singlet oxygen scavenger. Fluorescence lifetime(s) of PheSA in solution and when attached to the HA matrix has been monitored with ps resolved streak camera technology. Structural and fluorescence properties changes induced on HA-PheSA due to the presence of singlet oxygen were monitored using static light scattering (SLS), steady state fluorescence and ps time resolved fluorescence studies. SLS studies provided insight into the depolymerisation kinetics of PheSA derivatised HA matrix in the presence of singlet oxygen. Time resolved fluorescence studies grave insight into the dynamics of the reaction mechanisms induced on HA-PheSA by singlet oxygen. These studies provided insight into the medical relevance of PheSA derivatised HA: its capacity of scavenging singlet oxygen and of quenching PheSA fluorescence. These studies revealed that HA-PheSA is a strong quencher of electronic excited state PheSA and acts as a scavenger of singlet oxygen, thus medical applications of this derivatised form of HA may protect tissues and organs, such as skin, against reactive oxygen species damage.     

Histochemistry of skin glands of Trachycephalus aff. venulosus Laurenti, 1768 (Anura, Hylidae)

The skin of the adult tree frog, Trachycephalus aff. venulosus, was investigated by light microscopy. Histochemistry was carried out using various stain techniques and showed the basic integument morphology of the skin with squamous epithelium in the epidermis and connective tissue in the dermis, subdivided into spongy and compact layers. The integument observed contains two types of gland; the mucous glands and granular glands. The mucous glands are small and located in the upper layer of the stratum spongiosum of the connective tissue and contain neutral and acid sulfated glycoproteins. The granular glands are large and form a syncytial secretory compartment within the acinus, which is surrounded by smooth muscle cells. Histochemical analyses demonstrated the presence of collagen proteins in both types of gland. An unusual finding was the different densities of the granules in the granular glands. The morphological characteristics of the integument in Trachycephalus aff. venulosus was also compared with others species of Anura.     

细胞显微光谱研究竹红菌甲素在癌细胞内的滞留效应

利用细胞显微光谱技术,在20000～12000cm-1范围内测量了经过浓度为20μg*mL-1竹红菌甲素(HA)培养23个小时的小鼠淋巴癌细胞和未经HA培养的小鼠淋巴癌细胞的光谱.结果显示两者有明显的差异,说明HA使癌细胞发生了改变;在经HA培养的细胞光谱中还检测到HA的荧光峰,提示HA在癌细胞中滞留.发展了利用显微光谱研究细胞在药物作用下的变化这一技术.     

Hyaluronic Acid: Its Role in Voice

The extracellular matrix (ECM), once regarded simply as a structural scaffold, is now recognized as an important modulator of cellular behavior and function. One component that plays a prominent role in this process is hyaluronic acid (HA)--a molecule found in many different tissues. Research into the roles of HA indicates that it plays a key role in tissue viscosity, shock absorption, and space filling. Specifically, research into the role of HA in laryngology indicates that it has profound effects on the structure and viscosity of vocal folds. This article provides an introduction to the structure and biological functions of HA and its importance in voice. In addition, an overview of the pharmaceutical applications of HA is discussed.     

Dilated Minute Chambers in Laryngeal Vocal Fold Polyps: Histopathological and Ultrastructural Features

In Reinke's space of human vocal fold, type III collagen forms a three dimensional network and this contains numerous minute chambers in between these fibers. These compartments are occupied by glycosaminoglycans and glycoproteins. In laryngeal fold lesions, such as Reinke's edema and vocal fold polyps, proteoglycan (PG)/hyaluronic acid (HA) components of extracellular matrix increased. We investigated the size and quantity of the minute chambers within Reinke's space, filled with PG/HA with the aid of transmission electron microscopy. Eight vocal fold polyps and 10 mucosal biopsies (as control group) were all evaluated by light microscopy and electron microscopy. We detected that PG/HA in extracellular matrix had been increased in vocal fold lesions when compared with control group, by Alcian Blue-pH 2.5 stain. The mean volume of the chambers in Reinke's space of normal larynx was measured as 0.040233 µm² whereas the mean volume of these chambers in vocal fold polyps was measured as 6.420221 µm². The difference between the volumes of these chambers in vocal fold polyps and in control group was statistically significant (P = 0.001). Within these chambers PG/HA were found and PG/HA filling these chambers were increased in vocal fold polyps. We think proteoglycan and glycosaminoglycans, especially HA, play an important role in determining biochemical properties of vocal fold lesions.     

一种数字化高灵敏度荧光显微镜及其在竹红菌甲素研究中的应用

研制了一种可探测细胞微弱光图像的数字化高灵敏度荧光显微镜，采用视频数字化增强型ＣＣＤ系统作为高灵敏度的接收系统，像增强器亮度增益为４１，０００，因而这种荧光显微镜可探测到普通荧光显微镜不能观察的微弱光图像，并可减少荧光物质的浓度和激发光的强度，减少对细胞自然生理环境的影响，数字化高灵敏度荧光显微镜在给出细胞微弱荧光图像的同时，并可给出图像上每一像元的发光强度和细胞平均发光强度，使用此仪器已首次直接     

Osteoblast adhesion to clodronate-hydroxyapatite composite

The present study combined clodronate, one of bisphosphonates, to the surface of HA scaffold by chelation. The cell culture test and gene expression test were performed and evaluated the effect of clodronate modifying the HA on co-culturing osteoblast with HA scaffold in vitro. Characteristic peaks in XPS and FT-IR spectra indicated clodronate being immobilized on the surface of HA. The cell culture test indicated that the cells actively proliferated on the scaffolds. The results of RT-PCR showed there was no significant difference between two groups in expression of RANKL gene, while the expression of OPG gene showed higher expression in Clodronate-HA group. Clodronate had no obvious effect on the cytocompatibility of HA, and Clodronate-HA might be used as bone scaffold with potential ability to improve osteogenesis.     

Effect of surface modified hydroxyapatite on the tensile property improvement of HA/PLA composite

In this study, we modified the surface of hydroxyapatite (HA) particle by ring-opening polymerization of lactide (LA). The modified HA particles were characterized by IR and TGA. It was shown that LA could be graft-polymerized onto the surface of HA. A series of composites based on modified HA/PLA were further prepared and characterized. It indicated that the modified HA particles were well dispersed in PLA matrix than unmodified HA particles and the adhesion between HA particle and PLA matrix was improved. The modified HA/PLA composites showed good mechanical properties than that of unmodified HA/PLA.     

EPR investigations of structure of humic acids from compost, soil, peat and soft brown coal upon oxidation and metal uptake

Free radical concentration and theirg-values in humic acids (HA) isolated from various sources were studied by quantitative EPR technique. EPR data for HA formed during composting and natural humification processes occurring in soil, peat and brown coal are given. In more detail the EPR data were analyzed for brown coal HA under carbonization, air oxidation (150°C) as well as metal uptake (Ca(II), Zn(II), Cd(II), Hg(II), Co(II), Ni(II) and Cu(II)) and NO₂ reaction. Two groups of metal complexes were distinguished on the basis of their interaction with free radicals in HA. Ca(II), Zn(II), Cd(II) and Hg(II) ions increase free radical concentration, while Co(II), Ni(II) and Cu(II) ions quench the radicals compared to the raw HA. This phenomenon can be explained either by the strong interaction of the metal ions with active centres responsible for the quinone-hydroquinone-semiquinone equilibria, and/or by the antiferromagnetic interaction between radical spins and metal d orbitals. Gaseous ammonia was found to be a very useful base easily penetrating the solid matrix of HA and strongly influencing the equilibria. β-Diketone groups present in HA react with NO₂ yielding iminoxy radicals. In the HA-metal complexes these structural units are engaged in metal coordination which lowers effectiveness of the iminoxyl synthesis.     

阜新褐煤中腐植酸的提取

以阜新褐煤为原料,采用稀碱法提取腐植酸（HA）并通过红外光谱分析腐植酸的基本结构,为今后研究腐植酸的基本结构提供一定参考。实验主要考察了碱液浓度、反应温度、反应时间对腐植酸的提取率的影响,结果显示,用2.0%N aOH在50℃下反应75m in可得到最大提取率为13.696%。     

Cell types in the esophageal epithelium of Anguilla anguilla (Pisces, Teleostei). Cytochemical and ultrastructural characteristics

The esophagus is an important osmoregulatory organ in euryhaline teleost fishes; however, its histochemical and ultrastructural characteristics have been studied in few species. The structure of the esophageal mucosa was analyzed in the European eel (Anguilla anguilla L.) and compared to the findings reported for other species. The histological features of the esophageal mucosa, the histochemical characteristics of mucosecretory cells in the epithelial layer and the ultrastructural features of the different cell types in this layer in animals caught in fresh water and raised in the laboratory were analyzed. The role of mucus as a protective covering and its role in ion transport an the esophagus are discussed. In addition, comments on the role of tonofilament-rich epithelial cells in ion transport are made.     

XPS and bioactivity study of the bisphosphonate pamidronate adsorbed onto plasma sprayed hydroxyapatite coatings

This paper reports the use of X-ray photoelectron spectroscopy (XPS) to investigate bisphosphonate (BP) adsorption onto plasma sprayed hydroxyapatite (HA) coatings commonly used for orthopaedic implants. BPs exhibit high binding affinity for the calcium present in HA and hence can be adsorbed onto HA-coated implants to exploit their beneficial properties for improved bone growth at the implant interface. A rigorous XPS analysis of pamidronate, a commonly used nitrogenous BP, adsorbed onto plasma sprayed HA-coated cobalt-chromium substrates has been carried out, aimed at: (a) confirming the adsorption of this BP onto HA; (b) studying the BP diffusion profile in the HA coating by employing the technique of XPS depth profiling; (c) confirming the bioactivity of the adsorbed BP. XPS spectra of plasma sprayed HA-coated discs exposed to a 10 mM aqueous BP solution (pamidronate) for periods of 1, 2 and 24 h showed nitrogen and phosphorous photoelectron signals corresponding to the BP, confirming its adsorption onto the HA substrate. XPS depth profiling of the 2 h BP-exposed HA discs showed penetration of the BP into the HA matrix to depths of at least 260 nm. The bioactivity of the adsorbed BP was confirmed by the observed inhibition of osteoclast (bone resorbing) cell activity. In comparison to the HA sample, the HA sample with adsorbed BP exhibited a 25-fold decrease in primary osteoclast cells.     

Histochemical technique for the detection of chloride cells in fish

Chloride cells are responsible for ionic exchanges in the fish gill. These cells have been widely studied, considering its importance in vital functions of the gill, and because they proliferate when exposed to unfavorable environments. One of the main characteristic of these cells is an acidic cytoplasm, which has been used for identification through histochemical techniques with dyes such as Toluidine Blue and Hematoxylin and Eosin. However, these techniques can be problematic, since epithelial cells can, in certain situations, acquire acidic characteristics similar to those of chloride cells, thus staining in a similar way. Among other functions, chloride cells play a role in calcium uptake from the environment, and therefore have a high concentration of this element. Based on this information, this study aims at developing a specific protocol for the identification of chloride cells. With this purpose, the Von Kossa method specific for calcium was used combined with Hematoxylin counterstaining. Chloride cells had cytoplasm slightly stained with Hematoxylin and the presence of dark stained granules dispersed in the cytoplasm resulted from the Von Kossa reaction due to the calcium present in these cells. This was not found in any other gill cell. Thus, the technique used in this study was specific and efficient to identify chloride cells in fish gills.