Staining with Tetraphenylporphinesulfonate in Vivo

Tetraphenylporphinesulfonate (TPPS), a synthetic, nonnaturally occurring porphine derivative, was administered parenterally to tumor bearing rats and its in vivo localization was studied with fluorescence microscopy. TPPS was selectively localized in elastica and eosinophilic leukocytes, but not in other tissue sites rich in basic protein. The elastica of the aorta and medium sized arteries, as well as elastic fibers of the subendocardium, paratracheal connective tissue and bronchial walls showed the strongest red fluorescence. The intensity of fluorescence in these sites corresponded with the degree of sulfonation of the TPPS. The tumors showed moderate red fluorescence diffusely in the cytoplasm.     

Protein pattern of Xenopus laevis embryos grown in simulated microgravity

Numerous studies indicate that microgravity affects cell growth and differentiation in many living organisms, and various processes are modified when cells are placed under conditions of weightlessness. However, until now, there is no coherent explanation for these observations, and little information is available concerning the biomolecules involved. Our aim has been to investigate the protein pattern of Xenopus laevis embryos exposed to simulated microgravity during the first 6 days of development. A proteomic approach was applied to compare the protein profiles of Xenopus embryos developed in simulated microgravity and in normal conditions. Attention was focused on embryos that do not present visible malformations in order to investigate if weightlessness has effects at protein level in the absence of macroscopic alterations. The data presented strongly suggest that some of the major components of the cytoskeleton vary in such conditions. Three major findings are described for the first time: (i) the expression of important factors involved in the organization and stabilization of the cytoskeleton, such as Arp (actin-related protein) 3 and stathmin, is heavily affected by microgravity; (ii) the amount of the two major cytoskeletal proteins, actin and tubulin, do not change in such conditions; however, (iii) an increase in the tyrosine nitration of these two proteins can be detected. The data suggest that, in the absence of morphological alterations, simulated microgravity affects the intracellular movement system of cells by altering cytoskeletal proteins heavily involved in the regulation of cytoskeleton remodelling.     

Male reproduction cycle of Kulzer's Rock Lizard,Phoenicolacerta kulzeri (Müller & Wettstein, 1932), in Lebanon (Reptilia: Lacertidae)

The reproduction cycle of male Kulzer's Rock Lizard, Phoenicolacerta kulzeri, was studied in a mountain population living at 2000?m a.s.l. on Mount Sannine, Lebanon. Males showed active spermiogenesis in spring, following the renewal of the post hibernation activity, and in autumn, from September until they enter into hibernation in November. About 40% of males exhibited a short testicular regression period during the hottest months, in July and August. Relative testicular volume was correlated with male body size and varied seasonally. Males of P. kulzeri showed a more distinct reproductive pattern than the common reproductive pattern of most lacertid lizards in the Mediterranean region.     

Overexpression of Hp95 induces G1 phase arrest in confluent HeLa cells

Xp95, a protein recently identified in Xenopus laevis, is potentially involved in progesterone-induced Xenopus oocyte maturation. In this study, we cloned a human homologue of Xp95, designated Hp95, and examined the effect of its overexpression on the growth properties of human malignant HeLa cells which have lost the contact inhibition of cell proliferation. We observed that although HeLa cells did not undergo G1 phase arrest at any stage after confluence, they were able to downregulate their G1 phase CDK activities in response to confluence. When Hp95 was overexpressed in HeLa cells by transfection with a constitutive or an inducible expression vector containing a full-length Hp95 transgene, HeLa cells became able to undergo G1 phase arrest and form a monolayer culture after confluence. However, the G1 phase CDK activities in these Hp95 overexpressing cells were not inhibited further as compared to control cells after confluence. These results indicate that the defects in HeLa cells that cause the loss of contact inhibition of cell proliferation are in components downstream of the G1 phase CDKs and that overexpression of Hp95 counteracts some of these defects.     

A 36‐bp deletion in the alpha subunit of glutamate‐gated chloride channel contributes to abamectin resistance in Plutella xylostella

Diamondback moth (DBM), Plutella xylostella (L.) (Lepidoptera: Plutellidae), is one of the most destructive pests in Brassicaceae crops, such as Chinese cabbage (Brassica rapa L.). It is rapidly developing resistance to abamectin, the dominant insecticide utilized in controlling P. xylostella in China and other southeastern Asian countries. The target of abamectin, the alpha subunit of glutamate‐gated chloride channel (GluClα), is thought to be involved in the development of abamectin resistance in nematodes and insects. This study investigated variants of GluClα in both abamectin‐susceptible and resistant strains of P. xylostella. A comparison of the PxGluClα sequences revealed three variants, including a 63‐bp substitution, a 36‐bp deletion, and a 65‐bp insertion. The frequency of the 36‐bp deletion was much higher in the abamectin‐resistant strain compared to the susceptible strain, whereas the 63‐bp substitution and 65‐bp insertion showed no significant difference between the resistant and susceptible strains. The in vitro expression of PxGluClα (with or without the 36‐bp deletion) in Xenopus laevis (Daudin) oocytes indicated that PxGluClα with the 36‐bp deletion was less sensitive to both glutamate and abamectin compared to the wild‐type PxGluClα. These findings suggest that the variant 36‐bp deletion in PxGluClα may confer abamectin resistance in P. xylostella after continuous abamectin selection, providing new insights into the management of this pest and contributing to the development of new reagents for pest control.     

Dissection,Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation^1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and Müller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates ^12,14-18.While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells ^7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues ^{8,19-22,24-33}. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level ^{5,8,21,24,27-30,33-39}. Xenopus laevis, a classic model system for the study of early neural development ^{19,27,29,31-32,40-42}, serves as a particularly suitable system for retinal primary cell culture ^10,38,43-45.Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction ^25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products ^10,24,44-45.However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).     

南方红豆杉丛枝菌根(AM)的研究

研究了南方红豆杉根部丛枝菌根真菌(AMF)侵染情况、菌根形态结构以及根际土中AMF孢子的种类与数量.结果显示:南方红豆杉可与AMF形成典型的丛枝-泡囊型菌根,侵染率在71.2%~94.4%,但是历山、蟒河自然保护区的侵染强度优于人工栽培区;在南方红豆杉根际土中共分离鉴定出5种AMF,无梗囊霉属1种、球囊霉属4种,分别是:光壁无梗囊霉、地表球囊霉、地球囊霉、缩球囊霉、明球囊霉,其中光壁无梗囊霉为优势种;南方红豆杉的根由表皮、外皮层、内皮层、中柱组成,AMF只侵染表皮层、内皮层,不能侵染中柱.这为将来利用AMF接种技术进行南方红豆杉的繁殖、移植栽培以及紫杉醇的积累等研究提供了理论依据.     

Visual experience dependent regulation of neuronal structure and function by histone deacetylase 1 in developing Xenopus tectum in vivo

Histone deacetylase 1 (HDAC1) is thought to play pivotal roles in neurogenesis and neurodegeneration. However, the role of HDAC1 in neuronal growth and structural plasticity in the developing brain in vivo remains unclear. Here, we show that in the optic tectum of Xenopus laevis , HDAC1 knockdown dramatically decreased the frequency of AMPAR‐mediated synaptic currents and increased the frequency of GABAAR‐mediated currents, whereas HDAC1 overexpression significantly decreased the frequency of GABAAR‐mediated synaptic currents. Both HDAC1 knockdown and overexpression adversely affected dendritic arbor growth and visual experience‐dependent structural plasticity. Furthermore, HDAC1 knockdown decreased BDNF expression via a mechanism that involves acetylation of specific histone H4 residues at lysine K5. In particular, the deficits in dendritic growth and visually guided avoidance behavior in HDAC1‐knockdown tadpoles could be rescued by acute tectal infusion of BDNF. These results establish a relationship between HDAC1 expression, histone H4 modification and BDNF signaling in the visual‐experience dependent regulation of dendritic growth, structural plasticity and function in intact animals in vivo . © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 947–962, 2017