Label‐free quantitative proteome analysis of skeletal tissues under mechanical load

Skeletal tissue has the capability to adapt its mass and structure in response to mechanical stress. However, the molecular mechanism of bone and cartilage to respond to mechanical stress are not fully understood. A label‐free quantitative proteome approach was used for the first time to obtain a global perspective of the response of skeletal tissue to mechanical stress. Label‐free quantitative analysis of 1D‐PAGE‐LC/MS/MS based proteomics was applied to identify differentially expressed proteins. Differential expression analysis in the experimental groups and control group showed significant changes for 248 proteins including proteins related to proliferation, differentiation, regulation of signal transduction and energy metabolic pathways. Fluorescence labeling by incorporation of alizarin/calcein in newly formed bone minerals qualitatively demonstrated new bone formation. Skeletal tissues under mechanical load evoked marked new bone formation in comparison with the control group. Bone material apposition was evident. Our data suggest that 39 proteins were assigned a role in anabolic process. Comparisons of anabolic versus catabolic features of the proteomes show that 42 proteins were related to catabolic. In addition, some proteins were related to regulation of signal transduction and energy pathways, such as tropomyosin 4, fibronectin 1, and laminin, might be new molecular targets that are responsive to mechanical force. Differentially expressed proteins identified in this model may offer a useful starting point for elucidating novel aspects of the effects of mechanical force on skeletal tissue. J. Cell. Biochem. 108: 600–611, 2009. © 2009 Wiley‐Liss, Inc.     

Legionella pollution in cooling tower water of air-conditioning systems in Shanghai, China

Aims:  To determine Legionella pollution prevalence, describe the amount of Legionellae with respect to temperature in Shanghai cooling tower water (CTWs) in various types of public sites.
Methods and Results:  Six urban districts were selected as the study fields, adopting multiple-phase sampling methods. Routine culture was used to identify Legionellae. Of the samples, 58·9% (189/321) were observed to be positive, 19·9% were isolated over 100 CFU ml⁻¹. Legionella pneumophila serogroup 1 was the most frequently isolated species (155/189, 82·0%), followed by Leg. micdadei that was at the second place (44/189, 23·3%). The mean CFU ml⁻¹ of Legionellae in CTWs reached its peak from July to September. Over all 15·4% of the samples exceeding 100 CFU ml⁻¹ were observed in a hospital setting.
Conclusions:  The prevalence of Legionella pollution in CTWs, especially in CTWs of subway stations and hospitals, is worrying, and the positive rate and CFU ml⁻¹ of Legionellae in CTWs have a close relationship with air temperature.
Significance and Impact of the Study:  The study demonstrates pollution prevalence rates in different types of sites and various seasons, and provides a proportion of different serogroups of Legionellae . It illuminates an urgent need for dealing with the potential risk of legionellosis in Shanghai, through improved control and prevention strategies.     

The chloroplast protein RPH1 plays a role in the immune response of Arabidopsis to Phytophthora brassicae

Plant immune responses to pathogens are often associated with enhanced production of reactive oxygen species (ROS), known as the oxidative burst, and with rapid hypersensitive host cell death (the hypersensitive response, HR) at sites of attempted infection. It is generally accepted that the oxidative burst acts as a promotive signal for HR, and that HR is highly correlated with efficient disease resistance. We have identified the Arabidopsis mutant rph1 ( resistance to Phytophthora 1 ), which is susceptible to the oomycete pathogen Phytophthora brassicae despite rapid induction of HR. The susceptibility of rph1 was specific for P. brassicae and coincided with a reduced oxidative burst, a runaway cell-death response, and failure to properly activate the expression of defence-related genes. From these results, we conclude that, in the immune response to P. brassicae , (i) HR is not sufficient to stop the pathogen, (ii) HR initiation can occur in the absence of a major oxidative burst, (iii) the oxidative burst plays a role in limiting the spread of cell death, and (iv) RPH1 is a positive regulator of the P. brassicae -induced oxidative burst and enhanced expression of defence-related genes. Surprisingly, RPH1 encodes an evolutionary highly conserved chloroplast protein, indicating a function of this organelle in activation of a subset of immune reactions in response to P. brassicae . The disease resistance-related role of RPH1 was not limited to the Arabidopsis model system. Silencing of the potato homolog StRPH1 in a resistant potato cultivar caused susceptibility to the late blight pathogen Phytophthora infestans .     

Netrin-1诱导促性腺素释放激素神经元迁移的研究

目的:建立神经导向研究的模型,并鉴定netrin-1的神经导向功能.方法:用促性腺素释放激素(Gonadotropin-releasing hormone,GnRH)神经细胞为研究模型,建立双细胞团培养方法,用netrin-1诱导CmRH神经元亚克隆系NLT,观察细胞生长的诱导情况.结果:NLT细胞团在体外培养条件下,具有自发放射状迁移能力.在netrin-1诱导作用下,NLT细胞朝向netrin-1浓度梯度的方向生长,在靠近netrin-1细胞团的部位,netrin-1浓度梯度高,迁移出的细胞数目较多,约占总数目的68%,并且细胞迁移的平均距离较长为107.31μm;而在远离netrin-1细胞团部位,其netrin-1浓度梯度较低,迁移出的细胞数目相对较少约占32%,细胞迁移的平均距离较短为56.52μm.结论:netrin-1能诱导培养的NLT细胞发生明显的定向迁移,这为进一步深入研究netrin-1在神经系统中的功能和分子机制奠定了基础.     

A New Genetic Vaccine Platform Based on an Adeno-Associated Virus Isolated from a Rhesus Macaque

We created a hybrid adeno-associated virus (AAV) from two related rhesus macaque isolates, called AAVrh32.33, and evaluated it as a vaccine carrier for human immunodeficiency virus type 1 (HIV-1) and type A influenza virus antigens. The goal was to overcome the limitations of vaccines based on other AAVs, which generate dysfunctional T-cell responses and are inhibited by antibodies found in human sera. Injection of a Gag-expressing AAVrh32.33 vector into mice resulted in a high-quality CD8⁺ T-cell response. The resulting Gag-specific T cells express multiple cytokines at high levels, including interleukin-2, with many having memory phenotypes; a subsequent boost with an adenovirus vector yielded a brisk expansion of Gag-specific T cells. A priming dose of AAVrh32.33 led to high levels of Gag antibodies, which exceed levels found after injection of adenovirus vectors. Importantly, passive transfer of pooled human immunoglobulin into mice does not interfere with the efficacy of AAVrh32.33 expressing nucleoproteins from influenza virus, as measured by protection to a lethal dose of influenza virus, which is consistent with the very low seroprevalence to this virus in humans. Studies of macaques with vectors expressing gp140 from HIV-1 (i.e., with AAVrh32.33 as the prime and simian adenovirus type 24 as the boost) demonstrated results similar to those for mice with high-level and high-quality CD8⁺ T-cell responses to gp140 and high-titered neutralizing antibodies to homologous HIV-1. The biology of this novel AAV hybrid suggests that it should be a preferred genetic vaccine carrier, capable of generating robust T- and B-cell responses.The initial interest in vectors based on adeno-associated viruses (AAV) was for applications in gene therapy. Most of the initial work was with vectors derived from AAV serotype 2 (AAV2), which is one of the six initial isolates. In the first in vivo studies, several groups showed stable expression of the transgene Escherichia coli β-galactosidase following intramuscular (i.m.) injection of AAV2-LacZ without immune responses to the transgene (23, 44). The apparent tolerance of the host to AAV-encoded antigens to a variety of transgene products has been demonstrated in mice and some large animals (1, 35, 39). Several mechanisms have been proposed to explain the lack of T-cell responses following in vivo gene transfer with AAV, including ignorance (inadequate presentation of antigen), anergy, and suppression (1, 5, 18, 37).As applications of AAV vectors for in vivo gene transfer expanded, it became clear that the apparent immune privilege of AAV transgene products was not absolute. A number of examples emerged in which the host mounted vibrant T-cell responses to AAV-encoded transgene products. Several key parameters appeared to influence immunogenicity of the transgene. For example, Sarukhan et al. suggest that the subcellular localization of the protein influences the magnitude of the ensuing T-cell response after AAV gene transfer (37). The dose and route of administration of the AAV vector also contribute significantly to B- and T-cell responses to the transgene (3, 13). Wang et al. showed that inflammation at the site of AAV administration promotes antigen-specific immune responses to the transgene (47). A consistent observation has been that B-cell responses to AAV-encoded transgenes are much more intense and more consistently generated than CD8⁺ T-cell responses (8, 46, 51). A number of investigators have begun to explore AAV vectors as genetic vaccines against a variety of infectious and noninfectious diseases, based on the notion that it can be developed to stimulate transgene immune responses (14, 22, 26, 28, 48-50).The discovery of an expanded family of AAV capsids from human and nonhuman primates has provided an opportunity to evaluate the effects of capsid structure on vector performance. Most of this work has focused on the use of novel AAV serotypes for achieving higher levels of transgene expression for applications in gene therapy (7, 12, 36). Xin et al. recently evaluated, in mice, vectors as vaccines for human immunodeficiency virus type 1 (HIV-1) based on the original AAV isolates AAV1 to AAV6 and two novel AAVs we recently discovered, AAV7 and AAV8 (48). They showed significant capsid-dependent effects on T- and B-cell responses to HIV-1 gp160. We recently confirmed these observations and more thoroughly evaluated the quality of the CD8⁺ T-cell responses (26). AAV vectors of multiple serotypes encoding HIV-1 Gag were injected i.m. into mice, which all showed some level of CD8⁺ T-cell responses based on tetramer staining and peptide-induced gamma interferon (IFN-γ) expression. However, the quality of AAV-induced, Gag-specific T cells was substantially lower than that obtained with adenoviral vectors, based on several criteria. A majority of the tetramer-positive (Tet⁺) T cells were nonresponsive to antigen, and those that did respond to antigen produced low levels of IFN-γ and no interleukin-2 (IL-2). Very few memory T cells were generated, and animals primed with AAV vectors were not responsive to a boost with an adenoviral vector. However, all AAV serotypes studied did generate very high levels of antibodies to the Gag transgene product.A final issue to consider in the use of AAV as a genetic vaccine for HIV-1 is the presence of neutralizing antibodies (NAbs) to the vector due to prior AAV infections. We recently conducted an extensive screening of human populations from several continents and found high prevalence and high titers of NAbs to AAV1 and AAV2 and moderate levels of NAbs to AAV7 and -8 (4). In vivo gene transfer experiments indicate that AAV NAbs will likely impinge on vector efficacy (9, 33, 38).This study describes the creation of a novel AAV from rhesus macaque isolates, called AAVrh32.33, and its characterization as a genetic vaccine for HIV-1. AAVrh32.33 has properties unlike those of any others we have studied. We showed that vectors based on this novel capsid elicit strong CD8⁺ T-cell responses to reporter transgene products that are dependent on CD4⁺ T-cell help and dependent on signaling through CD40L and CD28 (L. E. Mays and J. M. Wilson, submitted for publication). Important to the use of this vector in the clinic is a very low incidence of NAbs to it in human populations. This study describes the development of vectors based on AAVrh32.33 as genetic vaccines.     

Reorganization of cytoskeleton during surfactant secretion in lung type II cells: a role of annexin II

The secretion of lung surfactant requires the movement of lamellar bodies to the plasma membrane through cytoskeletal barrier at the cell cortex. We hypothesized that the cortical cytoskeleton undergoes a transient disassembly/reassembly in the stimulated type II cells, therefore allowing lamellar bodies access to the plasma membrane. Stabilization of cytoskeleton with Jasplakinolinde (JAS), a cell permeable actin microfilament stabilizer, caused a dose-dependent inhibition of lung surfactant secretion stimulated by terbutaline. This inhibition was also observed in ATP-, phorbol 12-myristate 13-acetate (PMA)- or Ca(2+) ionophore A23187-stimulated surfactant secretion. Stimulation of type II cells with terbutaline exhibited a transient disassembly of filamentous actin (F-actin) as determined by staining with Oregon Green 488 Phalloidin. The protein kinase A inhibitor, H89, abolished the terbutaline-induced F-actin disassembly. Western blot analysis using anti-actin and anti-annexin II antibodies showed a transient increase of G-actin and annexin II in the Triton X-100 soluble fraction of terbutaline-stimulated type II cells. Furthermore, introduction of exogenous annexin II tetramer (AIIt) into permeabilized type II cells caused a disruption in the cortical actin. Treatment of type II cells with N-ethylmaleimide (NEM) resulted in a disruption of the cortical actin. NEM also inhibited annexin II's abilities to bundle F-actin. The results suggest that cytoskeleton undergoes reorganization in the stimulated type II cells, and annexin II tetramer plays a role in this process.     

Syntaxin 2 and SNAP-23 are required for regulated surfactant secretion

The secretion of lung surfactant in alveolar type II cells is a complex process involving the fusion of lamellar bodies with the plasma membrane. This process is somewhat different from the exocytosis of hormones and neurotransmitters. For example, it is a relatively slower process, and lamellar bodies are very large vesicles with a diameter of approximately 1 microm. SNARE proteins are the conserved molecular machinery of exocytosis in the majority of secretory cells. However, their involvement in surfactant secretion has not been reported. Here, we showed that syntaxin 2 and SNAP-23 are expressed in alveolar type II cells. Both proteins are associated with the plasma membrane, and to some degree with lamellar bodies. An antisense oligonucleotide complementary to syntaxin 2 decreased its mRNA and protein levels. The same oligonucleotide also inhibited surfactant secretion, independent of secretagogues. A peptide derived from the N-terminus of syntaxin 2 or the C-terminus of SNAP-23 significantly inhibited Ca(2+)- and GTPgammaS-stimulated surfactant secretion from permeabilized type II cells in a dose-dependent manner. Furthermore, introduction of anti-syntaxin 2 or anti-SNAP-23 antibodies into permeabilized type II cells also inhibited surfactant release. Our results suggest that syntaxin 2 and SNAP-23 are required for regulated surfactant secretion.