Dynamics of crowing development in the domestic Japanese quail (Coturnix coturnix japonica)

Species-specific behaviours gradually emerge, via incomplete patterns, to the final complete adult form. A classical example is birdsong, a learned behaviour ideally suited for studying the neural and molecular substrates of vocal learning. Young songbirds gradually transform primitive unstructured vocalizations (subsong, akin to human babbling) into complex, stereotyped sequences of syllables that constitute adult song. In comparison with birdsong, territorial and mating calls of vocal non-learner species are thought to exhibit little change during development. We revisited this issue using the crowing behaviour of domestic Japanese quail (Coturnix coturnix japonica). Crowing activity was continuously recorded in young males maintained in social isolation from the age of three weeks to four months. We observed developmental changes in crow structure, both the temporal and the spectral levels. Speed and trajectories of these developmental changes exhibited an unexpected high inter-individual variability. Mechanisms used by quails to transform sounds during ontogeny resemble those described in oscines during the sensorimotor phase of song learning. Studies on vocal non-learners could shed light on the specificity and evolution of vocal learning.     

三聚氰胺对花鲈的急性毒性实验研究

本文以花鲈(Lateolabrax japonicus)为实验对象,采用接触、腹腔注射及口服三种致毒方式,进行了非蛋白氮物质-三聚氰胺对花鲈的急性毒性实验研究。口服急性毒性实验中,通过在饲料中添加不同浓度的三聚氰胺（0、500、2000、5000及10000 mg/kg）,进行了21d的花鲈养殖实验。实验结果表明：三聚氰胺溶解度较低,其水溶液没有表现出急性毒性,LC50 > 3500 mg/L;而在腹腔注射致毒方式下,三聚氰胺对花鲈的半致死剂量LD50 = 890.07 mg/kg•w;LD5095%可信限为：778.63－1017.45 mg/kg•w;在口服急性毒性实验中,10000 mg/kg组三聚氰胺降低了花鲈的摄食与生长(p<0.05),饲料系数显著升高 (p<0.05);三聚氰胺对花鲈的存活率、肥满度、肝体比及脏体比均无显著影响 (p>0.05)。饲料中添加三聚氰胺没有显著影响花鲈血清的丙氨酸氨基转移酶、天冬氨酸氨基转移酶、总蛋白、葡萄糖、胆固醇、甘油三酯及尿素氮 (p>0.05),但显著影响了花鲈血清的碱性磷酸酶活性,10000 mg/kg组的碱性磷酸酶活性显著高于其他各处理组 (p<0.05),其他各组间无显著差异(p>0.05)。三聚氰胺对花鲈21d的最大未观察到有害作用剂量（NOAEL, no-observed-adverse-effect-level）为131.99 mg/kg•w•d。     

The impact of RNA interference of the subolesin and voraxin genes in male <Emphasis Type="Italic">Amblyomma hebraeum</Emphasis> (Acari: Ixodidae) on female engorgement and oviposition

Reducing or replacing the use of chemical pesticides for tick control is a desirable goal. The most promising approach would be to develop vaccines that protect hosts against tick infestation. Antigens suitable for the development of anti-tick vaccines will likely be those essential for vital physiological processes, and in particular those directly involved in feeding and reproduction. In this study genes from Amblyomma hebraeum Koch that encode for subolesin and voraxin were studied in male ticks by RNA interference (RNAi). Males (unfed or fed) were injected with dsRNA of (1) subolesin, (2) voraxin, (3) subolesin plus voraxin or (4) injection buffer, after which they were held off-host overnight and then allowed to feed on rabbits together with normal female A. hebraeum. Females that fed together with male ticks injected with subolesin or subolesin + voraxin dsRNA had a higher rate of mortality, weighed substantially less and produced a smaller egg mass than the controls. However, females feeding with males injected with voraxin dsRNA alone were not significantly different from the controls with respect to mortality, engorged weight or fecundity. However, as assessed by semi-quantitative RT-PCR, voraxin was not silenced in this study, the reasons for which remain unknown. The results of this study suggest that A. hebraeum subolesin is worthy of further testing as a candidate tick vaccine antigen.     

Molecular crowding affects diffusion and binding of nuclear proteins in heterochromatin and reveals the fractal organization of chromatin

The nucleus of eukaryotes is organized into functional compartments, the two most prominent being heterochromatin and nucleoli. These structures are highly enriched in DNA, proteins or RNA, and thus thought to be crowded. In vitro, molecular crowding induces volume exclusion, hinders diffusion and enhances association, but whether these effects are relevant in vivo remains unclear. Here, we establish that volume exclusion and diffusive hindrance occur in dense nuclear compartments by probing the diffusive behaviour of inert fluorescent tracers in living cells. We also demonstrate that chromatin‐interacting proteins remain transiently trapped in heterochromatin due to crowding induced enhanced affinity. The kinetic signatures of these crowding consequences allow us to derive a fractal model of chromatin organization, which explains why the dynamics of soluble nuclear proteins are affected independently of their size. This model further shows that the fractal architecture differs between heterochromatin and euchromatin, and predicts that chromatin proteins use different target‐search strategies in the two compartments. We propose that fractal crowding is a fundamental principle of nuclear organization, particularly of heterochromatin maintenance.     

PA‐MSHA inhibits proliferation and induces apoptosis through the up‐regulation and activation of caspases in the human breast cancer cell lines

To investigate the effects of PA‐MSHA (Pseudomonas aeruginosa‐mannose sensitive hemagglutinin) on inhibiting proliferation of breast cancer cell lines and to explore its mechanisms of action in human breast cancer cells. MCF‐10A, MCF‐7, MDA‐MB‐468, and MDA‐MB‐231HM cells were treated with PA‐MSHA or PA (Heat‐killed P. aeruginosa) at different concentrations and different times. Changes of cell super‐microstructure were observed by transmission electron microscopy. Cell cycle distribution and apoptosis induced by PA‐MSHA were measured by flow cytometry (FCM) with PI staining, ANNEXIN V‐FITC staining and Hoechst33258 staining under fluorescence microscopy. Western blot was used to evaluate the expression level of apoptosis‐related molecules. A time‐dependent and concentration‐dependent cytotoxic effect of PA‐MSHA was observed in MDA‐MB‐468 and MDA‐MB‐231HM cells but not in MCF‐10A or MCF‐7 cells. The advent of PA‐MSHA changed cell morphology, that is to say, increases in autophagosomes, and vacuoles in the cytoplasm could also be observed. FCM with PI staining, ANNEXIN V‐FITC and Hoechst33258 staining showed that the different concentrations of PA‐MSHA could all induce the apoptosis and G₀–G₁ cell cycle arrest of breast cancer cells. Cleaved caspase 3, 8, 9, and Fas protein expression levels were strongly associated with an increase in apoptosis of the breast cancer cells. There was a direct relationship with increased concentrations of PA‐MSHA but not of PA. Completely different from PA, PA‐MSHA may impart antiproliferative effects against breast cancer cells by inducing apoptosis mediated by at least a death receptor‐related cell apoptosis signal pathway, and affecting the cell cycle regulation machinery. J. Cell. Biochem. 108: 195–206, 2009. © 2009 Wiley‐Liss, Inc.     

Oral administration of Lactococcus lactis expressing Helicobacter pylori Cag7-ct383 protein induces systemic anti-Cag7 immune response in mice

To express the 3'-region (1152 bp) of the cag7 gene of Helicobacter pylori 51 strain, encoding the C-terminal 383 amino acid (ct383 aa) region of Cag7 protein that is known to cover the needle region of T4SS, in a live delivery vehicle Lactococcus lactis , the cag7-ct383 gene was amplified by PCR. DNA sequence analysis revealed that the amino acid sequence of Cag7-ct383 of H. pylori 51 shared 98.4% and 97.4% identity with H. pylori 26695 and J99, respectively. Intramuscular injection of the GST-Cag7-ct383 fusion protein into a rat could raise the anti-Cag7 antibody, indicating the immunogenicity of the Cag7-ct383 protein. When the cag7-ct383 gene was cloned in Escherichia coli–L. lactis shuttle vector (pMG36e) and transformed into L. lactis , the transformant could produce the Cag7-ct383 protein, as evidenced by Western blot analysis. The Cag7-ct383 protein level in the L. lactis transformant reached a maximum at the early stationary phase without extracellular secretion. The oral administration of the L. lactis transformant into mice generated anti-Cag7 antibody in serum in five of five mice. These results suggest that L. lactis transformant expressing Cag7-ct383 protein may be applicable as an oral vaccine to induce mucosal and systemic immunity to H. pylori .     

Oral vaccination of mice against Helicobacter pylori with recombinant Lactococcus lactis expressing urease subunit B

To determine whether a protective immune response could be elicited by oral delivery of a recombinant live bacterial vaccine, Helicobacter pylori urease subunit B (UreB) was expressed for extracellular expression in food-grade bacterium Lactococcus lactis . The UreB-producing strains were then administered orally to mice, and the immune response to UreB was examined. Orally vaccinated mice produced a significant UreB-specific serum immunoglobulin G (IgG) response. Specific anti-UreB IgA responses could be detected in the feces of mice immunized with the secreting lactococcal strain. Mice vaccinated orally were significantly protected against gastric Helicobacter infection following a challenge with H. pylori strain SS1. In conclusion, mucosal vaccination with L. lactis expressing UreB produced serum IgG and UreB-specific fecal IgA, and prevented gastric infection with H. pylori .     

Identification and characterization of novel recombinant vaccine antigens for immunization against genital Chlamydia trachomatis

Chlamydia trachomatis infection is the most common sexually transmitted bacterial infection worldwide, with over 91 million cases estimated annually. An effective subunit vaccine against Chlamydia may require a multivalent subunit cocktail of antigens in a single formulation for broad coverage of a heterogeneous major histocompatibility complex population. Herein, we describe the identification of novel C. trachomatis antigens by CD4+ and CD8+ T-cell expression cloning, serological expression cloning, and an in silico analysis of the C. trachomatis genome. These antigens elicited human CD4+ T-cell responses, and a subset proved to be immunogenic and protective when administered as immunoprophylactic vaccines against C. trachomatis challenge. Candidate vaccines consisting of the prioritized C. trachomatis antigens adjuvanted in a GlaxoSmithKline proprietary AS01B adjuvant were prioritized based on induction of solid protection against challenge in C57BL/6 and BALB/c mice with C. trachomatis . Some of the vaccines prevented bacterial shedding and colonization of the upper genital tract to varying degrees by mechanisms that may include CD4+ T cells.     

Analysis and stability of the Respiratory Syncytial Virus antigen in a T<Subscript>3</Subscript> generation of transgenic tomato plants

The integration, expression, and stability of the Respiratory Syncytial Virus (RSV)-F protein was analyzed in a T₃ generation of transgenic cherry tomato, Solanum lycopersicum L. cv. Swifty Belle, plants. Expression of the RSV-F antigen, under the control of the fruit-specific promoter E-8, was investigated in T₃ plants derived from a transgenic line, identified as #120. Transgene integration of the RSV-F gene in the T₃ generation was initially determined by polymerase chain reaction (PCR). PCR analysis from line 120-7-2 revealed that all T₃ plants were homozygous for the transgene; whereas, line 120-6-4 showed segregation for the transgene. Enzyme-linked immunosorbent assay (ELISA) was used to quantify levels of RSV-F protein in these plants, and protein levels ranged from 0–22 μg/g of fresh weight, with an average of ~3 μg/g fresh weight. Southern blot analysis of the highest expressing plants revealed presence of a single copy of the RSV-F transgene in these plants.