Genetic variability of resistance to Eimeria acervulina and E. tenella in chickens

Summary The genetic variability of 18 sire families of the Athens-Canadian randombred population infected with coccidiosis was assessed by examining the response variables of weight gain, packed red blood cell volume, mortality and coccidial lesions. A significant gain and PCV depression and high lesion scores for Eimeria tenella and E. acervulina were produced in the infected group compared to the noninfected group. Significant variation among the sire families was observed for all of the response variables except E. acervulina lesions and a significant sex x sire interaction was observed for weight gain. The heritability (h²) estimates for the response variables revealed that resistance to coccidiosis in chickens is moderately heritable. The h² estimates for gain and PCV increased with the coccidial infections indicating that maximum progress in selecting for resistance should be made when the population was exposed to coccidial infection. Gain was positively correlated to the other measures of resistance and thus selecting for coccidial resistance should not reduce growth rates. PCV was similarly correlated but had higher positive correlation with E. tenella lesion. Percent mortality which is the selection parameter in most coccidial selection programs was correlated with resistance to coccidiosis. The phenotypic and genotypic correlations demonstrated that chickens susceptible to E. tenella were also susceptible to E. acervulina. Total lesion scores were moderately to highly correlated with the other variables and would be a suitable variable to use in coccidiosis experimentation including a genetic selection program for resistance. This study shows that progress could be made in selecting for resistance to coccidiosis in chickens using one or a combination of these response variables.     

VLA-6 integrin distribution and calcium signalling in capacitated boar sperm.

Previous investigations showed that VLA-6 integrin present on boar sperm membrane can induce acrosome reaction upon exposure to laminin accumulated in expanded cumuli (Mattioli et al., 1998. To further investigate this novel sperm egg-recognition system, the authors studied the distribution of VLA-6 integrin on the membrane of boar sperm throughout capacitation and following acrosome reaction, and analyzed intracellular Ca(2+) changes occurring in spermatozoa exposed to laminin. Immunofluorescent localisation of VLA-6 revealed a low proportion (nearly 22%) of positive cells in freshly ejaculated sperm, with integrin mainly concentrated in clustered spots. After 3 hr incubation most of the spermatozoa showed integrin molecules on the membrane, with three different labeling patterns: fluorescence localised on the edge of the acrosome (58.2 +/- 14.2% of the cells); fluorescence uniformly spread over the whole sperm head (5.0 +/- 1.9%) and finally fluorescence concentrated in clustered spots (7.6 +/- 5.6%), as recorded in freshly ejaculated sperm. Twenty-nine percent of cells did not show any distinct fluorescence. Following acrosome reaction sperm with fluorescence on the acrosomal region virtually disappeared and the proportion of unstained cells rose from 29.2 +/- 9.2 to 69.0 +/- 10.1%. Electron microscopy demonstrated that VLA-6 integrin was exclusively located on the sperm membrane of intact spermatozoa. Confocal analysis showed that laminin triggers distinct Ca(2+) raises, and that sperm exposed and kept in the presence of laminin fully retained their ability to rise intracellular Ca(2+) in response to zona pellucida proteins. These data indicate that boar sperm accumulate VLA-6 integrin on the membrane and concentrate it on the acrosomal region as capacitation progresses. Probably due to this compartmentalisation, sperm exposed to laminin experience a Ca(2+) raise that originates in the anterior sperm head where it is more adequate for the induction of acrosome reaction. Mol. Reprod. Dev. 59:322-329, 2001.     

Specific lectins map the distribution of fibronectin and beta 1-integrin on living MDCK cells

The expression and dynamics of bound fibronectin and the sialylated integral membrane protein, beta 1-integrin, were analyzed on the apical membrane of living MDCK cells. Fibronectin was identified by its specific binding of fluorescent peanut agglutinin and sialylated beta 1-integrin by its binding of Sambucus nigra agglutinin. Confocal epifluorescence microscopy and laser scanning cytometry determined the distribution and abundance of binding sites of the two fluorescently labeled lectins. Both fibronectin and beta 1-integrin were restricted to specific regions uniformly distributed over the entire apical surface. Apical-surface fibronectin binding varied much more between cells than did the expression of beta 1-integrin. Sialylated beta 1-integrin colocalized >92% with membrane microplicae while fibronectin was unrelated to these surface structures. This lack of colocalization of the proteins was confirmed by double-labeling experiments. From the maturation dependence of the fibronectin-binding capacity and the differences in protein turnover times, it was evident that fibronectin did not bind to sialylated beta 1-integrin. Furthermore, desialylation of beta 1-integrin uncovered additional fibronectin receptors on the apical membrane. We conclude that these lectins permit tracking of two membrane-associated glycoproteins in living cells and that fibronectin binds only to desialylated beta 1-integrin on MDCK cells.     

Innocuousness and intracellular distribution of PKH67: A fluorescent probe for cell proliferation assessment

PKH dyes were initially developed by Horan et al. to provide appropriate probes for in vitro and in vivo cell tracking. It has been reported for many cell types that PKH bind irreversibly to the cell membrane without significantly affecting cell growth. Thus, these probes provide an opportunity for long-term cell monitoring and the identification of cells of interest among a heterogeneous cell population. An important feature is that upon cell division, the probe is partitioned equally between each daughter cell, making it possible to quantify tell fluorescence by flow cytometry. In this situation. the flow cytometric study of PKH67 characteristics shows that this probe does not affect the main cell-functions such as viability or proliferation. Moreover, the intracellular distribution of PKH67 is demonstrated by following its kinetics of internalization by confocal microscopy. These results present PKH67 as a probe suitable for dynamic analysis of cell proliferation as well as the study of intracellular localization and membrane recycling mechanisms.     

Effect of Bcl—2 and caspase—3 on calcium distribution in apoptosis of HL—60 cells

Apoptosis manifests in two major execution programs downstream of the death signal:the caspase pathway and organelle dysfunction.An important antiapoptosis factor,Bcl-2 protein,contributes in caspase pathway of apoptosis.Calcium,an important intracellular signal element in cells,is also observed to have changes during apoptosis,which maybe affected by Bcl-2 protein.We have previously reported that in Harringtonine (HT) induced apoptosis of HL-60 cells,there‘s change of intracellular calcium distribution,oving from cytoplast especially Golgi‘s apparatus to nucleus and accumulating there with the highest concentration.We report here that caspase-3 becomes activated in HT-induced apoptosis of HL-60 cells,which can be inhibited by overexpression of Bcl-2 protein.No sign of apoptosis or intracellular calcium movement from Golgi‘s apparatus to nucleus in HL-60 cells overexpressing Bcl-2 or treated with Ac-DEVD-CHO,a specific inhibitor of caspase-3.The results indicate that activated caspase-2 can promote the movement of intracellular calcium from Golgi‘s apparatus to nucleus,and the process is inhibited by Ac-DEVD-CHO(inhibitor of caspase-3),and that Bcl-2 can inhibit the movement and accumulation of intracellular calcium in nucleus through its inhibition on caspase-3.Calcium relocalization in apoptosis seems to be irreversible,which is different from the intracellular calcium changes caused by growth factor.     

The metareticulate pollen morphology of Alternanthera Forssk. (Gomphrenoideae,Amaranthaceae) and its taxonomic implications

Alternanthera (Amaranthaceae) is a diverse genus largely restricted to the American Tropics that belongs to the alternantheroid clade containing C₄ and C₃–C₄ intermediate species. This research focuses on the study of pollen characters by studying 13 species, representatives of the two major clades and subclades of Alternanthera. General palynological comparisons were conducted with light microscopy (LM), scanning electron microscopy (SEM) and with confocal laser scanning microscopy (CLSM) for exine ultrastructure. Twenty-five characters were measured and described for Alternanthera and among these, 14 pollen characters were used to discriminate pollen groups using cluster analysis and canonical analysis of principal coordinates (CAP). Pollen form and ornamentation, pores number, spines length, number of ektexinous bodies and nanospines on the ektexinous bodies on pore membranes, arrangement of nanopores and spines on structural elements, and metareticula form were taxonomically important and therefore used to construct the first palynological key to the alternantheroid clade species. Our study indicates that the seemingly subtle morphological variation of pollen is useful for recognising three main pollen types within Alternanthera. The much needed palynological terminology for describing the mesoporium in the metareticulate pollen of Amaranthaceae is provided.     

Immunohistochemical analysis of the distribution of type VI collagen in the lingual mucosa of rats during the morphogenesis of filiform papillae

Iwasaki, S., Yoshizawa, H. and Aoyagi, H. 2012. Immunohistochemical analysis of the distribution of type VI collagen in the lingual mucosa of rats during the morphogenesis of filiform papillae. —Acta Zoologica (Stockholm) 93 : 80–87. We examined the distribution after immunostaining of immunofluorescence of type VI collagen, differential interference contrast (DIC) images, and images obtained using confocal laser‐scanning microscopy in transmission mode, after toluidine blue staining, during morphogenesis of the filiform papillae, keratinization of the lingual epithelium and myogenesis in the rat tongue on semi‐ultrathin sections of epoxy resin‐embedded samples. Immunoreactivity specific for type VI collagen was dispersed over a relatively wide range of connective tissue in the mesenchyme of fetuses on day 15 after conception (E15), at which time the lingual epithelium was composed of one or two layers of cuboidal cells and the lingual muscle was barely recognizable. Slight immunoreactivity specific for type VI collagen was scattered within the lamina propria in fetuses on E17 and on E19, and immunoreactivity was relatively distinct on the connective tissue around the lingual muscle during myogenesis. In fetuses on E19, the epithelium was already stratified squamous. At postnatal stages from P0 to P14, keratinization of the lingual epithelium advanced gradually as morphogenesis of the filiform papillae proceeded during postnatal development. In newborns on P0, myogenesis of the tongue was almost completed. The intensity of immunoreactivity specific for type VI collagen at postnatal stages was mainly restricted on the endomysium and perimysium around the lingual muscle, while scant immunoreactivity was evident in the connective tissue in the lamina propria. Immunoreactivity around the fully mature lingual muscle on P7 and P14 was weaker than that on E19 and P0. Thus, type VI collagen appeared in the connective tissue that surrounded the lingual muscles such as the endomysium and perimysium, in parallel with changes in extracellular components during myogenesis of the tongue.     

Astrocytes and Neurons Express the Tight Junction-Specific Protein Occludin in Vitro

The expression of occludin, an integral plasma membrane protein specifically located at tight junctions, was studied in various epithelial and nonepithelial tissues by means of RT-PCR, Western blotting, and immunofluorescent staining. Besides detection in epithelial and endothelial tissue, expression of occludin was found in primary and secondary cultures of neurons and astrocytes. Differentiation of astrocytes in vitro led to a marked decrease in occludin expression. Extractability of occludin from plasma membranes differed considerably between epithelial and nonepithelial cells. Following treatment with Triton X-100, occludin was completely extracted from astrocytic membranes but not from membranes derived from MDCK cells, suggesting a difference in the cytoplasmic and/or plasma membrane anchoring of occludin between these cell types.     

Differential distribution of alpha-tubulin isotypes in Euplotes eurystomus determined by confocal immunofluorescence microscopy

Detergent permeabilized Euplotes eurystomus (a fresh water hypotrichous ciliate) was reacted with monoclonal and polyclonal antibodies specific for either detyrosinated or tyrosinated alpha-tubulin (Glu- or Tyr-tubulin). The isolated cytoskeleton-nuclear complex was examined by Western immunoblotting and by immunofluorescent and electron microscopic methods. Both Glu- and Tyr-tubulins were detected by immunoblot analysis. Immunofluorescent microscopy indicated that the alpha-tubulin isotypes are concentrated in different regions of permeabilized cells: Glu-tubulin is located primarily in cirri, membranelles, and surrounding the macro- and micronuclei. Tyr-tubulin is principally at the bases of cirri and membranelles. This differential distribution of alpha-tubulin isotypes is discussed in terms of current concepts concerning the correlation of tubulin post-translational modifications to microtubule stability. Confocal immunofluorescent imaging was of critical importance in clearly differentiating the Glu-tubulin isotype surrounding the macro- and micronuclei from a brilliantly fluorescent environment originating from cytoskeletal structures. In conjunction with conventional and stereo-electron microscopy, confocal optical microscopy provided convincing evidence for a "basket" of microtubules surrounding both nuclei.     

Eimeria acervulina and E. tenella:effect on methionine absorption by the avian intestine.

Absorption of l-methionine in the duodenum of intestine of chicks infected with Eimeria acervulina was markedly less than in uninoculated controls or birds infected with E. tenella. Absorption of methionine in the jejunal area of E. acervulina-infected birds was reduced although not as drastically as in the duodenum. There was no difference in the rate of methionine absorption by the ileum. The kinetics of methionine absorption showed that V_max (maximum velocity) in the duodenum and jejunum of E. acervulina-infected birds was reduced when compared with the V_max found in uninoculated controls or E. tenella-infected birds. There was no difference in the K_t (transport constant) regardless of the infection or the region of the intestine examined. No major consistent effect of decreased pH on the rate of methionine absorption could be shown.The broadly spatulate villi seen, using the scanning electron microscope, in control and E. tenella infected duodenum were absent in E. acervulina-infected duodenum. Instead, the villi were reduced in height and noticeably thickened. This reduction in villar height suggests that a portion of the reduction in methionine absorption was related to the change in surface area and loss of transport loci due to damage of the mucosal surface.     

激光生物学技术研究高脂血症大鼠红细胞变形能力改变及其机制

目的:通过研究红细胞膜流动性以及红细胞骨架结构的改变,进一步探讨高脂血症大鼠红细胞变形能力改变的机制。方法:16只Wistar大鼠随机分为两组:高血症组和对照组。高脂组给予高脂饮食。16周后,腹主动脉采血,采用酶比色法检测血浆甘油三脂、胆固醇含量;并利用激光衍射法测定红细胞变形指数、取向指数,荧光偏振法测定红细胞膜流动性,激光共聚焦显微镜观测红细胞骨架改变和红细胞F-actin的含量。结果:发现高脂血症大鼠红细胞的变形指数、取向指数以及红细胞膜的流动性显著降低(P<0.05),红细胞形态和骨架发生改变,F-actin含量显著降低(P<0.05)。结论:高脂血症大鼠红细胞变形能力降低与红细胞膜结构改变有一定的关系。     

Effects of biofilm structures on oxygen distribution and mass transport

Aerobic biofilms were found to have a complex structure consisting of microbial cell clusters (discrete aggregates of densely packed cells) and interstitial voids. The oxygen distribution was strongly correlated with these strutures. The voids facilitated oxygen transport from the bulk liquid through the biofilm, supplying approximately 50% of the total oxygen consumed by the cells. The mass transport rate from the bulk liquid is influenced by the biofilm structure; the observed exchange surface of the biofilm is twice that calculated for a simple planar geometry. The oxygen diffusion occurred in the direction normal to the cluster surfaces, the horizontal and vertical components of the oxygen gradients were of equal importance. Consequently, for calculations of mass transfer rates a three-dimensional model is necessary. These findings imply that to accurately describe biofilm activity, the relation between the arrangement of structural components and mass transfer must be undrstood. (c) 1994 John Wiley & Sons, Inc.     

Effect of pH on molecular constitution and distribution of hemoglobin in living erythrocyte

The molecular constitution of in situ hemoglobin (Hb) and their distribution in living erythrocyte were investigated versus pH using the technique of confocal Raman microscopy. Both Raman point spectra and line mapping measurements were performed on living erythrocytes in suspensions with pH values from 4.82 to 9.70. It was found that the Hb inside a living erythrocyte would dissociate into monomer/dimer when the cells are in low and high pH environments. In contrast to the homogeneous distribution of the Hbs in the cells in neutral suspension, there are more Hbs distributing around the cell membrane or binding to the membrane as pH increases. While in low pH, as the cell become spherical, most of the Hbs distribute to the central part of the cell. In summary, our investigation suggests that the variation of the external pH not only brings changes in the morphology and membrane structure of an erythrocyte, but also affects the constitution and distribution of its intracellular Hbs, thereby the flexibility of the cell membrane and the oxygenation ability of the Hb. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 348–354, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Simultaneous multi-parameter observation of Harringtonine-treating HL-60 cells with both two-photon and confocal laser scanning microscopy

Harringtonine (HT), a kind of anticancer drug isolated from Chinese herb-Cephalotaxus hainanensis Li, can induce apoptosis in promyelocytic leukemia HL-60 cells. With both two-photon laser scanning microscopy and confocal laser scanning microscopy in combination with the fluorescent probe Hoechst 33342, tetramethyrhodamine ethyl ester (TMRE) and Fluo 3-AM, we simultaneously observed HT-induced changes in nuclear morphology, mitochondrial membrane potential and intracellular calcium concentration ([Ca2+]i) in HL-60 cells, and developed a real-time, sensitive and invasive method for simultaneous multi-parameter observation of drug- treating living cells at the level of single cell.     

Flow cytometry and live confocal analysis for the evaluation of the uptake and intracellular distribution of FITC-ODN into HaCaT cells

In this study, the mechanism of the internalization and the cellular distribution of 59 fluorescein conjugated PS-ODN (FITC-ODN) after transfection with different mixed lipidic vesicles/oligo complexes (lipoplexes) have been investigated. Mixed lipidic vesicles were prepared with one of the most used cationic lipid (DOTAP) and different amounts of a cholic acid (UDCA) to release the oligo into HaCaT cells. Using flow cytometry, the cellular uptake of the oligo was studied with and without different inhibitors able to block selectively the different pathways involved in the internalization mechanism. The intracellular distribution of the oligo was analyzed by confocal laser scanning microscopy (CLSM), treating the cells with the lipoplexes and directly observing without any fixing procedure. To better carry out the colocalization studies, fluorescent-labeled markers, specific for the different cellular compartments, were coincubated with 59 fluorescein-conjugated 29-mer phosphorotioate oligonucleotide (FITC-ODN). The different lipidic vesicles affect the internalization mechanism of FITC-ODN. After using the inhibitors, the uptake of complexes involved a different internalization mechanism. The live CLSM analysis demonstrated that, after 1 hour from the complex incubation, the oligo was transferred into cells and localized into the endosomes; after 24 hours, the oligo was intracellularly localized close to the nuclear structure in a punctuate pattern. However, the results from fusion experiments showed also a binding of a quite low amount of oligo with the cell membranes.     

Glomerulus development in the absence of a set of mitral-like neurons in the insect olfactory lobe

Mitral cells are the first neurons in the mammalian olfactory bulb to synapse with olfactory receptor axons during glomerulus development, and in an invertebrate, the moth Manduca sexta, mitral-like neurons overlap very early with olfactory receptor axons as they begin to form protoglomeruli. The possibility for early interaction between receptor neurons and mitral-like neurons led us to ask whether such an interaction plays an essential role in glomerulus development. In the current study in the moth, we surgically removed a major class of these mitral-like neurons before glomeruli began to form and asked: (a) Is the formation of the array of olfactory glomeruli triggered by an interaction of the first-arriving receptor axons with the dendrites of mitral-like neurons? (b) At the level of individual glomeruli, must the mitral-like dendrites be in place either to maintain receptor axons in a glomerular arrangement, or to guide later-growing dendrites of other types into the developing glomeruli? Our results indicate that even without the participation of this group of mitral-like neurons, the array of sexually isomorphic ordinary glomeruli forms and the basic substructure of individual glomeruli develops apparently normally. We conclude that the mitral-like neurons in Manduca are not essential for the formation of ordinary olfactory glomeruli during development. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 41–52, 1998     

变应性鼻炎鼻分泌物中细胞内DNA和RNA代谢的观测

本文介绍应变性鼻炎鼻分泌物中细胞内DNA和RNA代谢变化的观测方法。以鼻分泌物中的多种炎性细胞为实验标本,经细胞贴壁、0.1%吖橙染色标记,37℃孵育15min;采用激光扫描共聚焦显微镜对细胞内DNA,RNA形态和含量进行观测。结果表明：本方法应用于变应性鼻炎鼻分泌物细胞内DNA、RNA代谢的研究是切实可行的。     

Vibrational spectroscopic imaging and live cell video microscopy for studying differentiation of primary human alveolar epithelial cells

Alveolar type II (ATII) cells in the peripheral human lung spontaneously differentiate toward ATI cells, thus enabling air‐blood barrier formation. Here, linear Raman and coherent anti‐Stokes Raman scattering (CARS) microscopy are applied to study cell differentiation of freshly isolated ATII cells. The Raman spectra can successfully be correlated with gradual morphological and molecular changes during cell differentiation. Alveolar surfactant rich vesicles in ATII cells are identified based on phospholipid vibrations, while ATI‐like cells are characterized by the absence of vesicular structures. Complementary, CARS microscopy allows for three‐dimensional visualization of lipid vesicles within ATII cells and their secretion, while hyperspectral CARS enables the distinction between cellular proteins and lipids according to their vibrational signatures. This study paves the path for further label‐free investigations of lung cells and the role of the pulmonary surfactant, thus also providing a basis for rational development of future lung therapeutics.