Concerning the dynamic instability of actin homolog ParM

Apoptosis is a highly conserved procedure of cell death and occurs under various stimuli, including oxidative stress. A small heat shock protein, alphaB-crystallin, is found to process resistance to apoptosis in some cells and tissues. But the mechanisms under this protective role are not fully understood. In the present study, we reported the early protective role of alphaB-crystallin against hydrogen peroxide-induced apoptosis in mice myogenic C(2)C(12) cells. alphaB-Crystallin interacted with p53, a proapoptotic protein, during cell apoptosis and such protein interaction mainly occurred in the cytoplasm of the cells, suggesting that the interaction of alphaB-crystallin with p53 might prevent the translocation of p53 from cytoplasm to mitochondria. Hence, this study provides a hint that alphaB-crystallin affects on p53 mitochondrial translocation during oxidative stress-induced apoptosis.     

Photomechanical wave-assisted molecular delivery in oral biofilms

Photomechanical waves (PW), the product of an intense light beam interaction with a target material, enhance molecular delivery across biological membranes and skin. The ability to deliver methylene blue (MB), a fluorescent probe and photosensitizer, into bacterial biofilms was demonstrated by applying PW on saliva-derived multi-species biofilms that were developed on agar surfaces in 24-well plates. PW were generated with a Q-switched Nd:YAG laser and were directed into the biofilms in the presence of 25 μg/ml MB. The biofilms were then irradiated with red light at 665 nm. After illumination, adherent bacteria were scraped and spread over the surface of blood agar plates. Survival fractions were calculated by counting bacterial colonies. Microbial analysis was performed via a colony lift method and a DNA checkerboard assay using whole genomic probes to 40 oral microorganisms. Visual analysis by confocal scanning laser microscopy demonstrated that the application of PW enhanced the penetration depth of MB in biofilms. Exposure to MB, PW and light led to a significant reduction of the mean levels of log₁₀ CFU counts compared with the group that received MB and light (P = 0.006). The DNA checkerboard assay showed some benefit from PW-assisted phototargeting in 25 biofilm microorganisms relative to phototreatment alone. Our data provide a basis for further exploration and optimization of PW parameters for complete eradication of microorganisms in oral microcosm biofilms.     

Molecular sequencing and analysis of soluble methane monooxygenase gene clusters from methanotroph <Emphasis Type="Italic">Methylomonas</Emphasis> sp<Emphasis Type="Italic">.</Emphasis> GYJ3

Summary A methanotroph Methylomonas sp. GYJ3 was isolated, whose sMMO genes and 16S rDNA were sequenced and analysed, demonstrating that the bacterium might be a type I methanotroph (γ-Proteobacteria) and was closer to Methylomonas sp. KSWIII/KSPIII. This result was consistent with the result previously determined by biochemistry and morphological taxonomy. Sequence comparison among six open reading frames and the deduced amino acid sequences of the sMMO genes from six strains revealed that the strain GYJ3 had highly conserved regions in MMOX with other strains, amounting to 78–99% homology at protein level and 71–97% homology at DNA level. Highly conserved sequences lay in two iron-binding regions. Furthermore, scanning electron microscopy of the strain GYJ3 showed rod shapes with a slightly bent configuration on the even surfaces and with plump bodies.     

Early chloroplastic alterations analysed by optical coherence tomography during a harpin-induced hypersensitive response

The hypersensitive response has been mostly studied by molecular and biochemical methods after sample destruction. The development of imaging techniques allows the monitoring of physiological changes before any signs of cell death. Here, we follow the early steps of a hypersensitive-like response induced by the bacterial elicitor harpin in Nicotiana sp. We describe cytological modifications after inoculation of the harpin protein, using confocal fluorescence microscopy (CFM) and optical coherence tomography (OCT), an interferometric-based microscopy. The changes detected by CFM occurred 5 h after harpin infiltration and corresponded to a redistribution of the chloroplasts from the upper to the inner regions of the palisade mesophyll cells which could be related to a perturbation in the microtubule network. Using OCT, we were able to detect a decrease in chloroplast backscattered signal as early as 30 min after harpin infiltration. A simple physical model, which accounted for the structure and distribution of thylakoid membranes, suggested that this loss of scattering could be associated with a modification in the refractive index of the thylakoid membranes. Our OCT observations were correlated with a decrease in photosynthesis, emphasizing changes in chloroplast structure as one of the earliest hallmarks of plant hypersensitive cell death.     

流行性感冒裂解疫苗免疫原性试验及电镜观察

实验报告了以三硝基甲苯X-100(Triton X-100)为裂解剂对流行性感冒病毒裂解效果的电镜观察结果及裂解疫苗的免疫原性。通过对病毒纯化、裂解、再纯化制备的3批流行性感冒裂解疫苗样品的电镜观察,表明使用此裂解剂能使疫苗中的病毒裂解完全,无完整病毒颗粒,裂解效果好。安全试验和免疫试验结果表明疫苗的安全性好,免疫效果好。     

牛磺酸对乳鼠心肌细胞内钙浓度的影响

研究低氧、复氧对乳鼠心肌细胞内钙离子浓度的影响,以及牛磺酸在模拟心肌缺血/再灌注(I/R)过程中对细胞内钙的调节作用。采用SD大鼠乳鼠进行心肌细胞培养,建立模拟I/R模型。以Fluo-4/AM荧光指示剂负载,应用激光共聚焦显微镜技术(confocal laser scanning microscope,CLSM)检测心肌细胞钙离子浓度的变化。对照组心肌细胞内钙离子荧光强度(23.71±2.37U)较低;低氧180 min后复氧即刻,钙离子荧光强度开始增加(57.52±8.31U),复氧180 min后钙离子荧光强度(71.13±4.74U)显著增高(P<0.01vs对照组)。而牛磺酸组细胞内钙离子荧光强度较模拟I/R组显著降低[(42.42±4.17U)vs(71.13±4.74U),P<0.01]。心肌细胞缺血/缺氧导致Ca2+超载;模拟I/R Ca2+超载加剧,而牛磺酸有明显减轻心肌细胞模拟I/R时Ca2+超载的作用。     

Imaging and manipulating the structural machinery of living cells on the micro- and nanoscale

The structure, physiology, and fate of living cells are all highly sensitive to mechanical forces in the cellular microenvironment, including stresses and strains that originate from encounters with the extracellular matrix (ECM), blood and other flowing materials, and neighbouring cells. This relationship between context and physiology bears tremendous implications for the design of cellular micro-or nanotechnologies, since any attempt to control cell behavior in a device must provide the appropriate physical microenvironment for the desired cell behavior. Cells sense, process, and respond to biophysical cues in their environment through a set of integrated, multi-scale structural complexes that span length scales from single molecules to tens of microns, including small clusters of force-sensing molecules at the cell surface, micron-sized cell-ECM focal adhesion complexes, and the cytoskeleton that permeates and defines the entire cell. This review focuses on several key technologies that have recently been developed or adapted for the study of the dynamics of structural micro-and nanosystems in living cells and how these systems contribute to spatially-and temporally-controlled changes in cellular structure and mechanics. We begin by discussing subcellular laser ablation, which permits the precise incision of nanoscale structural elements in living cells in order to discern their mechanical properties and contributions to cell structure. We then discuss fluorescence recovery after photobleaching and fluorescent speckle microscopy, two live-cell fluorescence imaging methods that enable quantitative measurement of the binding and transport properties of specific proteins in the cell. Finally, we discuss methods to manipulate cellular structural networks by engineering the extracellular environment, including microfabrication of ECM distributions of defined geometry and microdevices designed to measure cellular traction forces at micron-scale resolution. Together, these methods form a powerful arsenal that is already adding significantly to our understanding of the nanoscale architecture and mechanics of living cells and may contribute to the rational design of new cellular micro-and nanotechnologies.     

Substance P and acetylcholine are co-localized in the pathway mediating mucociliary activity in Rana pipiens

Mucociliary activity is an important clearance mechanism in the respiratory system of air breathing vertebrates. Substance P (SP) and acetylcholine play a key role in the stimulation of the mucociliary transport in the frog palate. In this study, retrograde neuronal tracing was combined with immunocytochemistry for SP and choline acetyl transferase (ChAT) in the trigeminal ganglion and for neurokinin-1 receptor (NK1R) in the palate of Rana pipiens. The cells of origin of the palatine nerve were identified in the trigeminal ganglion using the retrograde tracer Fluorogold (FG). Optimal labeling of FG cells in the trigeminal ganglion was obtained at 96 h of exposure. Immunoflorescent shows that SP and acetylcholine are co-localized in 92% of the cells labeled with FG in the trigeminal ganglion. NK1 receptors were found in the membrane of epithelial and goblet cells of the palate. Ultrastructural study of the palate showed axonal-like endings with vesicles in connection with epithelial and goblet cells. These results further support the concerted action of both neurotransmitters in the regulation of mucociliary activity in the frog palate.     

Ultrastructural analysis reveals striking differences of intercellular contact lengths in bovine embryos produced in vivo, in vitro and by somatic cell nuclear transfer

Cellular coherence and communication, thus cell-to-cell contact is an indispensable premise to sustain the formation of complex, multi-cellular organisms. We have analyzed intercellular contact lengths in NT-cloned bovine embryos compared to the in vivo or in vitro produced counterparts. Therefore, ultrastructural analysis was carried out by transmission electron microscopy (TEM) at the 8-cell and blastocyst stage of development. To obtain embryos generated in vivo, oviducts of superovulated cows were flushed 3 days after insemination, subsequent to slaughter. Standard in vitro maturation (IVM) and -fertilization (IVF) were utilized to obtain in vitro embryos. Cloned embryos by somatic nuclear transfer were produced by the handmade cloning (HMC) procedure. The points of apposition/focal contact points (CPs) between the blastomeres were of the shortest order in cloned embryos (236 +/- 135 nm) and of highest order in the in vivo produced embryos (2,085 +/- 1,540 nm), although no significant differences regarding the blastomere sizes in the various groups of 8-cell embryos could be established. In summary, the CP lengths in case of in vitro and in vivo 8-cell embryos were, on an average, five or nine times longer, respectively, than in the case of the cloned embryos. These differences of CP lengths vanished in embryos reaching the blastocyst stage of embryonic development in all the three groups of embryos. The observed differences of intercellular contact length at distinct stages of embryonic development could be responsible for differences in intercellular communication between the blastomeres at the beginning of cellular differentiation. These may be one reason for the lower developmental competence of cloned (NT) embryos.     

Equine zona protein synthesis and ZP structure during folliculogenesis, oocyte maturation, and embryogenesis

In the equine, the zona pellucida (ZP) is the major barrier to successful in vitro fertilization. Therefore the aim of our studies was to analyze species-specific features of the equine ZP in regard to structure and glycoprotein ZPB and ZPC expression sites during oocyte development and embryogenesis. The equine ZP revealed high immunological cross-reactivity to porcine ZPB and ZPC. In the ovary, the distribution of ZPB and ZPC was co-localized and correlated with the developmental stage of the follicle. ZPB and ZPC expression started in the oocyte of the late primordial and primary follicle. In the secondary follicle, both the oocyte and the cumulus cells contributed to ZPB and ZPC synthesis. After in vivo maturation the oocyte stopped ZPB and ZPC production whereas the cumulus cells continued synthesis. Contrary, in vitro matured (IVM) cumulus-oocyte-complexes (COCs) revealed a reverse expression pattern. This was correlated to alterations in the distribution, number, and size of pores in the ZP. In the zona, N-acetylglucosamine residues were co-localized with ZPC. The acellular glycoprotein capsule surrounding early equine embryos was negative for ZPB and ZPC. Our results imply that in the horse ZPB and ZPC glycoprotein expression is differentially regulated during folliculogenesis, oocyte maturation, and embryogenesis. Contrary to the bovine and porcine, zona protein synthesis during in vivo maturation is completely overtaken by the cumulus cells implying that in the horse these cells are crucial for zona integrity. During IVM, the cumulus cells lose their ability to synthesize glycoproteins leading to alterations in the zona structure.