Primary administration of Lactobacillus johnsonii NCC533 in weaning period suppresses the elevation of proinflammatory cytokines and CD86 gene expressions in skin lesions in NC/Nga mice

The administration of probiotic lactic acid bacteria (LAB) has been studied for its potential to prevent atopic dermatitis (AD). The objective of this study was to assess the inhibitory mechanism of a skin lesion by LAB using an experimental model that we previously demonstrated in NC/Nga mice. Lactobacillus johnsonii NCC533 (La1) was administered orally to the La1 group from 20 to 22 days after birth, while phosphate-buffered saline was given to the control group. After the induction of skin lesions in 6-week-old mice, the expression of genes supposedly involved in AD was evaluated. Gene expression of the proinflammatory cytokines [interleukin-8 (IL-8), IL-12 and IL-23] was significantly enhanced in the lesional skin of the control group by the induction of the lesion, whereas gene expression of those in the La1 group was not elevated. Interestingly, expression of the costimulatory molecule CD86 showed a pattern similar to the expression of the cytokines in the lesional skin. Moreover, the La1 group showed a significantly lower gene expression of CD86 in Peyer's patches and mesenteric lymph nodes than the control group. The suppression of proinflammatory cytokines and CD86 by primary administration of La1 may significantly contribute to the inhibitory effect on the skin lesion.     

Rb transport in Leishmania infantum promastigotes under various in vitro culture conditions

The survival of Leishmania, which encounter drastic changes of environment during their life-cycle, requires regulation and control of ionic concentrations within the cell. We analysed the influence of growth stage, ionic composition of the medium, heat and acidic stress on ⁸⁶Rb⁺ influx in L. infantum promastigetes. Proliferating promastigotes exibited faster and higher ⁸⁶Rb⁺ uptake than stationary cells. Cl⁻ anion did not have any effect, but in the presence of physiological concentration of HCO₃⁻, ⁸⁶Rb⁺ uptake was significantly increased. This enhancing effect was only partially inhibited by N,N′-dicyclohexylcarbodiimide (DCCD), a blocker of ion-translocating ATPases. ⁸⁶Rb⁺ influx was abolished by N-ethylmaleimide (NEM), indicating a major contribution of plasma membrane transporters. Heat shock and acidic shock notably decreased ⁸⁶Rb⁺ influx. Our data provide indirect evidence that an energy-dependent system which brings K⁺ in, such as K⁺/H⁺-ATPase evidenced by Jiang et al. (1994), is active in Leishmania in different environments. Mechanism(s) other than ion-translocating ATPase occur, at least in the presence of HCO₃⁻, and their contribution to K⁺ pathways varies in different environmental conditions.     

十字花科植物CYP86MF基因同源序列的克隆与进化分析

为了从分子水平阐明十字花科植物间的亲缘进化关系,给植物种质资源的创建提供理论依据,试验根据课题组已报道的CYP86MF基因编码的氨基酸保守区域设计特异引物,运用PCR技术分别从十字花科6个属11个物种中分离克隆到了CYP86MF基因的同源序列,经比较分析,结果表明：这些同源序列的相似性达80%以上,所推导的氨基酸序列相似性达70%以上,且两者种间差异分别为1.0% ~ 5.7%和2.6% ~ 7.3%,属间差异分别是5.6% ~ 22.5%和7.3% ~ 31.2%;由氨基酸序列构建的分子系统树可知,在亲缘进化关系上芸薹属与萝卜属较近,其他依次为蔊菜属、拟南芥属、荠菜属,而与诸葛菜属最远。因此,CYP86MF基因的核苷酸及其可能编码的氨基酸序列差异属间较种间大,它可用于属间的分类等级研究,而难用于属以下的分类等级研究。     

A better understanding of the role of the CTLA–CD80/86 axis in the treatment of autoimmune diseases

Autoimmune diseases are diseases in which the regulatory mechanisms of the immune response are disturbed. As a result, the body loses self-tolerance. Since one of the main regulatory mechanisms of the immune response is the CTLA4–CD80/86 axis, this hypothesis suggests that autoimmune diseases potentially share a similar molecular basis of pathogenesis. Hence, investigating the CTLA4–CD80/86 axis may be helpful in finding an appropriate treatment strategy. Therefore, this study aims to investigate the molecular basis of the CTLA4–CD80/86 axis in the regulation of the immune response, and then its role in developing some autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, and multiple sclerosis. As well, the main therapeutic strategies affecting the CTLA4–CD80/86 axis have been summarized to highlight the importance of this axis in management of autoimmune diseases.     

Inhibition of Human Cytomegalovirus Infection by IE86-Specific Short Hairpin RNA-Mediated RNA Interference

To develop a gene therapeutic method for human cytomegalovirus (HCMV), the IE86 specific short hairpin (sh) RNA expressing vector was constructed and subsequently transfected into MRC-5 cells. After infection of these cells with HCMV AD169, expression of IE86 was reduced strikingly as compared to the control. In addition, the inhibitory effect corresponded to a decrease in viral DNA replication and the virus-induced cytopathic effect. Measurement of the virus yield demonstrated that infection of cells expressing IE86-specific shRNA resulted in suppression of the formation of infectious viral progeny. These observations indicate that IE86 can be used as an effective target against HCMV infection using RNA interference (RNAi) technology, which provides new possibilities for anti-HCMV studies.     

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported¹. Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases². Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells³. This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis^3,4. Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.     

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.     

Methods to Explore the Influence of Top-down Visual Processes on Motor Behavior

Kinesthetic awareness is important to successfully navigate the environment. When we interact with our daily surroundings, some aspects of movement are deliberately planned, while others spontaneously occur below conscious awareness. The deliberate component of this dichotomy has been studied extensively in several contexts, while the spontaneous component remains largely under-explored. Moreover, how perceptual processes modulate these movement classes is still unclear. In particular, a currently debated issue is whether the visuomotor system is governed by the spatial percept produced by a visual illusion or whether it is not affected by the illusion and is governed instead by the veridical percept. Bistable percepts such as 3D depth inversion illusions (DIIs) provide an excellent context to study such interactions and balance, particularly when used in combination with reach-to-grasp movements. In this study, a methodology is developed that uses a DII to clarify the role of top-down processes on motor action, particularly exploring how reaches toward a target on a DII are affected in both deliberate and spontaneous movement domains.     

Fasting-induced dissociation of cationic and secretory events in pancreatic islets

In pancreatic islets removed from 48 h-fasted rats, as distinct from fed animals, the release of insulin evoked by D-glucose is more severely impaired than that evoked by 2-ketoisocaproate. This decreased secretory response to D-glucose contrasts with an unimpaired cationic response to the sugar in terms of the glucose-induced decrease in both 86Rb and 45Ca outflow from pre-labelled islets. Likewise, fasting only causes a modest decrease of the secondary rise in 45Ca outflow evoked by D-glucose in islets perifused at normal Ca2+ concentration. The latter decrease appears more marked, however, if the cationic response to glucose is expressed relative to that evoked by 2-ketoisocaproate in islets removed from rats in the same nutritional state. It is concluded that, in the process of nutrient-stimulated insulin release, neither the decrease in K+ conductance (inhibition of 86Rb outflow) nor the sequestration of Ca2+ by intracellular organelles and/or direct inhibition of Ca2+ outward transport (decrease in 45Ca outflow) represent the sole determinant(s) of the subsequent gating of Ca2+ channels (secondary rise in 45Ca efflux).