Proteomic analysis provides new insight into the chicken eggshell cuticle

The cuticle is the outermost layer of the avian eggshell, whose protein constituents remain virtually unknown. We hypothesize that cuticle components play a major role in microbial resistance, since eggs with incomplete or absent cuticle are more susceptible to bacterial contamination. In this study we extracted proteins from the outermost non-calcified layer of the cuticle of chicken eggs and subjected them to LC/MS/MS proteomic analysis. We identified 47 cuticle proteins with high confidence and reproducibility. Two proteins, similar to Kunitz-like protease inhibitor and ovocalyxin-32 (a carboxypeptidase A inhibitor), were the most abundant of the cuticle proteins. A number of proteins known to have antimicrobial activity in the egg were detected (lysozyme C, ovotransferrin, ovocalyxin-32, cystatin, ovoinhibitor) as well as possible new candidates (myeloperoxidase, ovocalyxin-36 and members of the SERPIN family). This is the first comprehensive report of cuticle proteome, a starting point to determine cuticle function and the molecular basis of its antimicrobial properties.     

Flow-cytometric analysis of chicken red blood cells.

Flow-cytometric analysis of acriflavin-Feulgen stained chicken erythrocytes shows a complex distribution of amounts of deoxyribonucleic acid fluorescence, the profile consisting of a main peak and a right hand shoulder. This bimodal distribution, an artifact characteristically seen on analysis of flattened cells using orthogonal flow systems, results from fluorescence emission in preferred directions stemming from the combined effects of refractility and orientation of the cells. The shoulder disappears on analysis of lysed erythrocyte ghosts, also on analysis of cells in a medium whose refractive index approximates that the cells. An orientation effect for matrue erythrocytes was indicated by reanalysis of fractions after sorting on the basis of high and low fluorescence or scatter signals. Both fractions gave the original range of values on reanalysis, although some changes in shape of the profile and in the peak positions for the sorted cells were seen. Sodium dodecyl sulfate treatment of stained cells "loosened" the cells' structure, yielding lowered scatter values, and fluorescence values approaching those of the shoulder. The average fluorescence emission of the erythrocytes was lower than that of reticulocytes and lymphocytes. The values of the latter correspond closely, although coincidently, to that the erythrocyte shoulder values. Dual parameter analysis of forward light scatter, and fluorescence, which was detected at 90 degrees to the laser beam, showed the low fluorescence to be accompanied by low scatter signal, and the high fluorescence among the cells with the high scatter signal. The lowered forward scatter signal is due to a wider scattering of light from cells oriented edge-on to the detector, and loss of signal beyond the acceptance angle of the detector. These results suggest that the preferred directions for fluorescence are in the plane of the cells, and the values are dependent on the cells' orientation in the stream. These interpretations were supported by the results of analysis of partially oriented cells. The approaches used and conclusions arrived at are similar to those of Gledhill et al (16), Van Dilla et al (37), in their analysis of fluorescence of flat sperm cells although the affects in the case of the erythrocytes are less extreme.     

A biomimetic motility assay provides insight into the mechanism of actin-based motility

Abiomimetic motility assay is used to analyze the mechanism of force production by site-directed polymerization of actin. Polystyrene microspheres, functionalized in a controlled fashion by the N-WASP protein, the ubiquitous activator of Arp2/3 complex, undergo actin-based propulsion in a medium that consists of five pure proteins. We have analyzed the dependence of velocity on N-WASP surface density, on the concentration of capping protein, and on external force. Movement was not slowed down by increasing the diameter of the beads (0.2 to 3 microm) nor by increasing the viscosity of the medium by 10(5)-fold. This important result shows that forces due to actin polymerization are balanced by internal forces due to transient attachment of filament ends at the surface. These forces are greater than the viscous drag. Using Alexa488-labeled Arp2/3, we show that Arp2/3 is incorporated in the actin tail like G-actin by barbed end branching of filaments at the bead surface, not by side branching, and that filaments are more densely branched upon increasing gelsolin concentration. These data support models in which the rates of filament branching and capping control velocity, and autocatalytic branching of filament ends, rather than filament nucleation, occurs at the particle surface.     

Nucleus-anchoring cytoskeleton in chicken red blood cells

Cytoskeleton of chicken erythrocytes was studies studied after extraction of the cells with Triton X-100. In phase contrast microscopy the extracted cells were seen as ghost-like structures with preserved morphology, distinct nucleus and surrounding plasma membrane remnant. In electron microscopy, dense matrix-like nucleus, fibrillar plasma membrane residue and filaments, ca. 10 nm in diameter, traversing the cytoplasmic domain, were seen. Distinct bands of molecular weight 68000, 53000, 32000 and bands of both higher and lower molecular weight were obtained by polyacrylamide gel electrophoresis of the extracted cells. These results indicate that intermediate filaments, forming the nucleus-anchoring cytoskeleton, are present in nucleated chicken erythrocytes as part of cellular cytoskeleton.     

超低温保存鸡红细胞条件的选择

通过比较液氮冻存鸡红细胞(CRBC)时,不同组成的冻存保护液及不同的降温程序,对CRBC活力的影响来选择最佳的冻存条件。结果表明,含15％二甲基亚砜(DMSO)的不添加血清RP—MI-1640培养液．具有良好保持冻存CRBC活力的作用。以样品悬置液氮罐口10min→浸入液氮的程序降温,经40℃水浴复苏,CRBC的活力高于90％。CRBC由于廉价易得,是学生人数较多时进行细胞冻存与复苏实验的优良材料。     

Functional analysis of SLO2 provides new insight into the role of plant PPR proteins

PPR (Pentatricopeptide repeat) proteins are mainly involved in RNA metabolism. In Arabidopsis, the PPR family is composed of more than 450 members; however, only few of them were functionally characterized. In a previous report,1 we identified a novel mitochondrial PPR RNA editing factor, named SLO2, which is responsible for 7 editing events in Arabidopsis. Loss-of-function mutation in SLO2 results in plant growth retardation, and delayed development, and leads to the dysfunction of mitochondrial complex I, III and IV. slo2 is the first example of a single gene mutation affecting 3 complexes of the mitochondrial electron transport chain. This Short Communication discusses the conservation of upstream regions of editing sites affected by SLO2 and illustrates the effect of mutation of SLO2 on activation of the alternative pathway. We also reflect upon the implications and perspectives of these findings.     

Anion transport in embryonic and adult chicken red blood cells

The rates of Cl and SO₄ transport at 0° and 37°C, respectively, have been measured under exchange conditions for red blood cells of embryonic and adult chickens. It was found that the rate of self-exchange of SO₄ in embryonic red cells decreases as the embryo matures, and that the SO₄ transport rate was lower in adult compared to embryonic red cells. In contrast, no difference in the rate of Cl self-exchange was found between adult and embryonic red cells.     

The amphioxus genome provides unique insight into the evolution of immunity

Immune systems evolve as essential strategies to maintain homeostasis with the environment, prevent microbial assault and recycle damaged host tissues. The immune system is composed of two components, innate and adaptive immunity. The former is common to all animals while the latter consists of a vertebrate-specific system that relies on somatically derived lymphocytes and is associated with near limitless genetic diversity as well as long-term memory. Deuterostome invertebrates provide a view of immune repertoires in phyla that immediately predate the origins of vertebrates. Genomic studies in amphioxus, a cephalochordate, have revealed homologs of genes encoding most innate immune receptors found in vertebrates; however, many of the gene families have undergone dramatic expansions, greatly increasing the innate immune repertoire. In addition, domain-swapping accounts for the innovation of new predicted pathways of receptor function. In both amphioxus and Ciona, a urochordate, the VCBPs (variable region containing chitin-binding proteins), which consist of immunoglobulin V (variable) and chitin binding domains, mediate recognition through the V domains. The V domains of VCBPs in amphioxus exhibit high levels of allelic complexity that presumably relate to functional specificity. Various features of the amphioxus immune repertoire reflect novel selective pressures, which likely have resulted in innovative strategies. Functional genomic studies underscore the value of amphioxus as a model for studying innate immunity and may help reveal how unique relationships between innate immune receptors and both pathogens and symbionts factored in the evolution of adaptive immune systems.     

A novel statin-mediated “prenylation block-and-release” assay provides insight into the membrane targeting mechanisms of small GTPases

Ras super-family small GTPases regulate diverse cellular processes such as vesicular transport and signal transduction. Critical to these activities is the ability of these proteins to target to specific intracellular membranes. To allow association with membranes Ras-related GTPases are post-translationally modified by covalent attachment of prenyl groups to conserved cysteine residues at or near their C-terminus. Here we used the HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase (HMGCR) inhibitor mevastatin to develop a ‘prenylation block-and-release’ assay that allows membrane targeting of prenylated proteins to be visualized in living cells. Using this assay we investigated the cytosol to membrane targeting of several small GTPases to compartments of the secretory and endocytic pathways. We found that all Rabs tested were targeted directly to the membrane on which they reside at steady-state and not via an intermediate location as reported for Ras and Rho proteins. However, we observed that the kinetics of cytosol to membrane targeting differed for each Rab tested. Comparison of the mevastatin sensitivity and kinetics of membrane targeting of Rab23, Rab23 prenylation motif mutants and H-Ras revealed that these parameters are strongly dependent upon the prenyl transferase with Rab geranylgeranyl transferase substrates exhibiting higher sensitivity and requiring greater time to recover from mevastatin inhibition than farnesyl transferase substrates. We propose that this assay is a useful tool to investigate the kinetics, biological functions and the mechanisms of membrane targeting of prenylated proteins.     

A species-specific qPCR assay provides novel insight into range expansion of the Mediterranean monk seal (Monachus monachus) by means of eDNA analysis

Biodiversity and Conservation - The monk seal is the most endangered pinniped worldwide and the only one found in the Mediterranean, where its distribution and abundance have suffered a drastic...     

Lipopolysaccharide-induced suppression of antibody-directed cell-mediated cytotoxicity to chicken red blood cells

Prior treatment with commercially prepared and acetone-extracted lipopolysaccharide (LPS) was found to suppress the expression of antibody-directed, cell-mediated cytotoxicity (ADCC) to chicken red blood cells (CRBC) by spleen cells from C57/BL10, C3H, and BALB/c mice. The in vitro incubation with commercial LPS suppressed ADCC-CRBC activity of spleen cells from both C3H/HeJ and C3H/HeN mice. Only the C3H/HeN strain was suppressed when treated with purified LPS. ADCC-CRBC activity of neonatal spleen cells could be suppressed after a 3-hr in vivo incubation with LPS while adult spleen cells required a minimum of 15 hr preincubation.     

人多能干细胞向红细胞的诱导分化

目前,体外生成人红细胞的实验技术较为复杂,为优化诱导步骤,采用两步法将人多能干细胞诱导分化为红细胞。首先,利用人多能干细胞(包括Rh阴性A型的脐带间充质干细胞(hUCMSC~(Rh-A))和人iPS(hiPS)细胞)在BVF培养液中进行诱导分化得到CD31~~+和CD34~+的阳性细胞群。经PCR和流式细胞仪检测CD31和CD34的表达发现,hUCMSC~(Rh-A)细胞诱导得到的CD31和CD34阳性细胞率分别是5.3%和22.7%;hiPS细胞诱导得到的CD31和CD34阳性细胞率分别是31.2%和8.2%。第二步,将获得的CD31~+和CD34~+的阳性细胞群在多种生长因子的作用下经过36 d诱导,分化为成熟红细胞。经吉姆萨染色检测得到的红细胞在形态和大小上与正常人红细胞相近,且存在血细胞去核的现象。荧光定量RT-PCR检测到了globin的表达,其中β-globin的表达量占20%以上。将得到的红细胞收集到离心管中,自然沉降后可见红色的红细胞沉淀。上述研究为大量制备人红细胞提供了新的有效的技术方法。