CodY在单核细胞增生李斯特菌运动和毒力方面的作用

目的:探究全局性转录调控因子CodY在单核细胞增生李斯特菌(Listeria monocytogenes,Lm)鞭毛运动和细菌毒力方面的作用。方法:通过同源重组的方法敲除Lm染色体上CodY的编码基因codY并成功构建缺失菌株的回复菌株;利用平板泳动法观测鞭毛运动的变化,RT-qPCR检测与鞭毛运动相关基因的转录表达;比较野生型菌株EGDe与CodY缺失菌株对细菌溶血活性、棉铃虫幼虫的半致死剂量和主要的毒力因子LLO和毒力基因调控蛋白PrfA转录表达的影响。结果:同野生型菌株相比,CodY缺失菌株鞭毛运动和相关基因,以及主要的毒力因子LLO和PrfA的转录表达显著降低(P≤0.01),溶血活性显著降低(P≤0.01),对棉铃虫幼虫的半致死剂量上升了5.8倍。结论:CodY在Lm鞭毛运动和细菌毒力调控方面具有重要作用。     

Reactive oxygen species as mediators of sperm capacitation and pathological damage

Oxidative stress plays a major role in the life and death of mammalian spermatozoa. These gametes are professional generators of reactive oxygen species (ROS), which appear to derive from three potential sources: sperm mitochondria, cytosolic L‐amino acid oxidases, and plasma membrane Nicotinamide adenine dinucleotide phosphate oxidases. The oxidative stress created via these sources appears to play a significant role in driving the physiological changes associated with sperm capacitation through the stimulation of a cyclic adenosine monophosphate/Protein kinase A phosphorylation cascade, including the activation of Extracellular signal regulated kinase‐like proteins, massive up‐regulation of tyrosine phosphorylation in the sperm tail, as well as the induction of sterol oxidation. When generated in excess, however, ROS can induce lipid peroxidation that, in turn, disrupts membrane characteristics that are critical for the maintenance of sperm function, including the capacity to fertilize an egg. Furthermore, the lipid aldehydes generated as a consequence of lipid peroxidation bind to proteins in the mitochondrial electron transport chain, triggering yet more ROS generation in a self‐perpetuating cycle. The high levels of oxidative stress created as a result of this process ultimately damage the DNA in the sperm nucleus; indeed, DNA damage in the male germ line appears to be predominantly induced oxidatively, reflecting the vulnerability of these cells to such stress. Extensive evaluation of antioxidants that protect the spermatozoa against oxidative stress while permitting the normal reduction‐oxidation regulation of sperm capacitation is therefore currently being undertaken, and has already proven efficacious in animal models.     

Persistent mechanical stretch-induced calcium overload and MAPK signal activation contributed to SCF reduction in colonic smooth muscle in vivo and in vitro

Gastrointestinal (GI) distention is a common pathological characteristic in most GI motility disorders (GMDs), however, their detail mechanism remains unknown. In this study, we focused on Ca²⁺ overload of smooth muscle, which is an early intracellular reaction to stretch, and its downstream MAPK signaling and also reduction of SCF in vivo and in vitro. We successfully established colonic dilation mouse model by keeping incomplete colon obstruction for 8 days. The results showed that persistent colonic dilation clearly induced Ca²⁺ overload and activated all the three MAPK family members including JNK, ERK and p38 in smooth muscle tissues. Similar results were obtained from dilated colon of patients with Hirschsprung's disease and stretched primary mouse colonic smooth muscle cells (SMCs). Furthermore, we demonstrated that persistent stretch-induced Ca²⁺ overload was originated from extracellular Ca²⁺ influx and endoplasmic reticulum (ER) Ca²⁺ release identified by treating with different Ca²⁺ channel blockers, and was responsible for the persistent activation of MAPK signaling and SCF reduction in colonic SMCs. Our results suggested that Ca²⁺ overload caused by smooth muscle stretch led to persistent activation of MAPK signaling which might contribute to the decrease of SCF and development of the GMDs.     

Histone deacetylase 6 and cytoplasmic linker protein 170 function together to regulate the motility of pancreatic cancer cells

Pancreatic cancer is a devastating disease with the worst prognosis among all the major human malignancies. The propensity to rapidly metastasize contributes significantly to the highly aggressive feature of pancreatic cancer. The molecular mechanisms underlying this remain elusive, and proteins involved in the control of pancreatic cancer cell motility are not fully characterized. In this study, we find that histone deacetylase 6 (HDAC6), a member of the class II HDAC family, is highly expressed at both protein and mRNA levels in human pancreatic cancer tissues. HDAC6 does not obviously affect pancreatic cancer cell proliferation or cell cycle progression. Instead, it significantly promotes the motility of pancreatic cancer cells. Further studies reveal that HDAC6 interacts with cytoplasmic linker protein 170 (CLIP-170) and that these two proteins function together to stimulate the migration of pancreatic cancer cells. These findings provide mechanistic insight into the progression of pancreatic cancer and suggest HDAC6 as a potential target for the management of this malignancy.     

膜联蛋白A2对人肝癌细胞生物学行为的影响

为了研究膜联蛋白A2(ANXA2)基因表达水平与人肝癌细胞生物学行为之间的关系,采用已优化转染条件的磷酸钙法将针对ANXA2的siRNA重组质粒导入人肝癌细胞SMMC-7721并观察对靶基因表达及细胞生物学行为的影响。以半定量RT-PCR和Western blotting检测转染后不同时间(24 h、48 h、72 h和96 h)ANXA2 mRNA及蛋白表达变化;以MTT法、Hoechast33258染色及体外损伤修复实验等分析抑制ANXA2表达后对SMMC-7721细胞生物学行为的影响。结果显示:所设计的四条siRNA序列均不同程度抑制了ANXA2 的表达(p<0.05),且呈时间依赖性;细胞生长能力及运动能力被显著抑制(p<0.05),其抑制效应与ANXA2 表达敲低程度呈正相关。结论:ANXA2的高表达水平与肝癌细胞的凋亡、生长及运动能力密切相关,以RNAi方法抑制该基因的表达可以有效地抑制肝癌细胞的恶性生物学行为。上述结果为肝癌的实验性基因治疗研究提供了新的思路和对策。     

Selection of a high quality subpopulation of frozen thawed bovine spermatozoa by filtration in a Sephadex column. An assessment of functional effects of progesterone

It is accepted that cryopreservation exerts deleterious effects on functional characteristics of mammalian spermatozoa. Conventional procedures for processing frozen-thawed gametes, such as centrifugation, produce additional damage. In the present work, we investigated the efficacy of processing bovine cryopreserved semen by filtration in a Sephadex column (SF group) or by washing by centrifugation (100 g, 10 min, twice) (W group); the results obtained from both procedures were compared to untreated samples (C group). The effects of in vitro addition of progesterone (10 μM, 20 min) upon sperm functional activity were studied also. The evaluated sperm parameters were concentration, motility (progressive or non progressive cells), viability and acrosome reaction. They were measured at time 0 (immediately after processing) or after 4 h incubation in capacitating conditions. Sperm concentration was (× 10⁻⁶): 37.5 ± 5.4 in C, 8.3 ± 2.1 in W and 12.5 ± 2.9 in SF. The percentages of motile, progressive, viable or acrosome intact gametes were significantly higher in SF than in W or in C. in SF group, after 4 h incubation in capacitating conditions, progesterone increased significantly the population of acrosome reacted cells whereas this parameter was not modified when the cells were incubated in absence of heparin. Motility and viability were not modified by the hormone. We conclude that Sephadex filtration method is an adequate tool to obtain a subpopulation of spermatozoa with superior quality, as assessed by motility, viability and acrosomal integrity; besides, our results strongly support that, as in other species, progesterone would be a physiological inductor of acrosome reaction in bovine.     

Sperm ultrastructure of five species of the Neotropical ant genus Pseudomyrmex (Hymenoptera: Formicidae)

The seminal vesicles of adult males of five species of Pseudomyrmex were prepared for light and transmission electron microscopy. The Pseudomyrmex spermatozoa are long and slender with similar morphology. The head region has an acrosome and a nucleus. In all the studied species, two morphologically distinct types of acrosomal vesicles were observed, a long structure, as observed in all known ants, and a pear‐shaped one, never before observed in ants. The nucleus is elongated and both condensed and loose chromatin are present. The flagellum has an axoneme, a centriolar adjunct, two mitochondrial derivatives and two accessory bodies. The centriolar, the mitochondrial derivatives and the accessory bodies are similar to observations in most ant species that have been studied. The axoneme presents an uncommon 9 + 9 + 1 microtubule arrangement and the central microtubule has 13 protofilaments. The acrosomal dimorphism and the different levels of chromatin organization are exclusive characteristics of Pseudomyrmex. Furthermore, the 9 + 9 + 1 microtubule arrangement is different from all Hymenoptera, as well as from most insects, which present a 9 + 9 + 2 arrangement. These new morphological characters that are specific to Pseudomyrmex, are valuable synapomorphies of the genus and can be used in taxonomic characterization of the Pseudomyrmecinae subfamily and in phylogenetic analyses in Formicidae family.     

Sperm ultrastructure in representatives of six bivalve families from Peter the Great Bay, Sea of Japan

The ultrastructure of sperm in seven species of bivalves, the representatives of six families, Arcidae (Anadara broughtonii, Arca boucardi), Anomiidae (Pododesmus macrochisma), Tellinidae (Macoma tokyoensis), Ostreidae (Crassostrea gigas), Myidae (Mya japonica) and Trapezidae (Trapezium liratum) is described. All the studied sperm were typical tail sperm, adapted to external insemination, which, however, had a specific structure. Differences were revealed in the form of head, acrosome structure and number of mitochondria. The studied species of the above families had their specific morphology, the Arcidae species had a bullet- or barrel-shaped head with four or five mitochondria in the middle part; the Anomiidae had conic head, the acrosome with periacrosome material and four mitochondria (a basic feature of sperm is the axial core entering periacrosome material and consisting of bundle of actin filaments); the Myidae had a curved conic head and four mitochondria; in the Tellinidae the head was bullet-shaped, the periacrosome material contained a fibril component and four mitochondria; the Trapezidae had sperm of a conic form with spherical acrosome. The spherical sperm of C. gigas were similar to sperm of Saccostrea commercialis and Crassostrea virginica, but with some distinctions in the acrosome substructure. The morphology of sperm testified to the correct attribution of the Crassostreidae family as a synonym to the Ostreidae family.     

Biomimetic membrane systems to study cellular organization

During many cellular processes such as cell division, polarization and motility, the plasma membrane does not only represent a passive physical barrier, but also provides a highly dynamic platform for the interplay between lipids, membrane binding proteins and cytoskeletal elements. Even though many regulators of these interactions are known, their mutual interdependence appears to be highly complex and difficult to study in a living cell. Over the past few years, in vitro studies on membrane–cytoskeleton interactions using biomimetic membranes turned out to be extremely helpful to get better mechanistic insight into the dynamics of these processes. In this review, we discuss some of the recent developments using in vitro assays to dissect the role of the players involved: lipids in the membrane, proteins binding to membranes and proteins binding to membrane proteins. We also summarize advantages and disadvantages of supported lipid bilayers as model membrane.