siRNA-mediated silencing of CD44 delivered by Jet Pei enhanced Doxorubicin chemo sensitivity and altered miRNA expression in human breast cancer cell line (MDA-MB468)

Molecular Biology Reports - CD44, as a superficial cellular glycoprotein, is an essential factor in cell–cell and cell–matrix interaction. The CD44 expression level has been...     

Effects of Toxoplasma gondii and Toxocara canis Antigens on WEHI-164 Fibrosarcoma Growth in a Mouse Model

Cancer is the main cause of death in developed countries. However, in underdeveloped countries infections and parasitic diseases are the main causes of death. There are raising scientific evidences indicating that parasitic infections induce antitumor activity against certain types of cancers. In this study, the effects of Toxoplasma gondii and Toxocara canis egg antigens in comparison with Bacillus Calmette Guerin (BCG) (known to have anticancer distinctive) on WEHI-164 fibosarcoma transplanted to BALB/c mice was investigated. Groups of 6 male BALB/c mice injected with T. gondii antigen, BCG, or T. canis egg antigen as case groups and alum alone as control groups. All mice were then challenged with WEHI-164 fibrosarcoma cells. The mice were examined for growth of the solid tumor and the tumor sizes were measured every other day up to 4 wk. The mean tumor area in T. gondii, BCG, or alum alone injected mice in 4 different days of measurements was 25 mm², 23 mm², and 186 mm² respectively. Also the mean tumor area in T. canis injected mice in 4 different days was 25.5 mm² compared to the control group (alum treated) which was 155 mm². T. gondii parasites and T. canis egg antigens induced inhibition of the tumor growth in the fibrosarcoma mouse model. We need further study to clarify the mechanisms of anti-cancer effects.     

人HCN4通道的滞后现象:影响窦房结起搏的潜在决定因素(英文)

超极化活化环核苷酸门控(hyperpolarization-activated cyclic-nucleotide-gated,HCN)通道参与调制心脏跳动的节律和速率。与HCN1和HCN2有所不同,慢通道HCN4可能不存在电压依赖的滞后现象。本研究采用单细胞膜片钳方法,在稳定转染hHCN4的HEK293细胞上进行电生理记录,观察hHCN4通道是否存在滞后现象,以及cAMP对其的调制作用;同时采用实时定量RT-PCR方法检测窦房结和心房组织中HCNs的表达。电压钳实验结果显示hHCN4电流(Ih)激活随着保持电位超极化的变化而向去极化方向移动。三角电位变化钳(triangular ramp)和动作电位钳的结果也显示了hHCN4的滞后现象。cAMP增加Ih电流幅度,且使电流激活向去极化方向移动,从而改变内源性hHCN4滞后行为。RT-PCR结果显示,人窦房结组织主要表达HCN4,占75%,HCN1占21%,HCN2占3%,HCN3占0.7%。以上结果提示,人窦房结组织主要表达HCN4亚型,hHCN4的Ih存在电压依赖性的滞后现象,且受cAMP调制。由此推断,hHCN4通道的滞后现象可能在窦房结起搏活动中起到了关键作用。     

The Cellulase KORRIGAN Is Part of the Cellulose Synthase Complex

Plant growth and organ formation depend on the oriented deposition of load-bearing cellulose microfibrils in the cell wall. Cellulose is synthesized by a large relative molecular weight cellulose synthase complex (CSC), which comprises at least three distinct cellulose synthases. Cellulose synthesis in plants or bacteria also requires the activity of an endo-1,4-β-d-glucanase, the exact function of which in the synthesis process is not known. Here, we show, to our knowledge for the first time, that a leaky mutation in the Arabidopsis (Arabidopsis thaliana) membrane-bound endo-1,4-β-d-glucanase KORRIGAN1 (KOR1) not only caused reduced CSC movement in the plasma membrane but also a reduced cellulose synthesis inhibitor-induced accumulation of CSCs in intracellular compartments. This suggests a role for KOR1 both in the synthesis of cellulose microfibrils and in the intracellular trafficking of CSCs. Next, we used a multidisciplinary approach, including live cell imaging, gel filtration chromatography analysis, split ubiquitin assays in yeast (Saccharomyces cerevisiae NMY51), and bimolecular fluorescence complementation, to show that, in contrast to previous observations, KOR1 is an integral part of the primary cell wall CSC in the plasma membrane.Cellulose microfibrils are synthesized by a hexameric multiprotein complex at the plasma membrane called the cellulose synthase complex (CSC). Genetic analysis, expression data, and coimmunoprecipitation experiments have demonstrated that a functional CSC contains at least three different nonredundant cellulose synthase (CESA) isoforms (Höfte et al., 2007). CESA1, CESA3, and CESA6-like are involved in cellulose biosynthesis during primary cell wall deposition, whereas CESA4, CESA7, and CESA8 are essential for cellulose synthesis in the secondary cell wall (Taylor et al., 1999, 2000, 2003; Desprez et al., 2007; Persson et al., 2007). CSCs labeled by fluorescently tagged CESA proteins migrate in the plasma membrane along cortical microtubules (CMTs), propelled by the polymerization of the β-1,4-glucans (Paredez et al., 2006). Partial depolymerization of CMTs using oryzalin showed that the organized trajectories of CSCs depend on the presence of an intact CMT array. The CSC-microtubule interaction is mediated at least in part by a large protein, POMPOM2/CELLULOSE SYNTHASE INTERACTING1, that binds to both CESAs and microtubules (Lei et al., 2014). Interestingly, complete depolymerization of CMTs does not alter the velocity of the complexes, illustrating that CMTs are necessary for the guidance of CSCs but not for their movement (Paredez et al., 2006). The microtubule cytoskeleton also has a role in the secretion and internalization of CSCs (Crowell et al., 2009; Gutierrez et al., 2009)KORRIGAN1 (KOR1) is a membrane-bound endo-1,4-β-d-glucanase (EGase) that is also required for cellulose synthesis (Nicol et al., 1998). Enzymatic analysis of a recombinant and soluble form of the Brassica napus KOR1 homolog showed substrate specificity for low-substituted carboxymethyl cellulose and amorphous cellulose but no activity on crystalline cellulose, xyloglucans, or short cellulose oligomers (Mølhøj et al., 2001; Master et al., 2004). Fractionation of microsomes demonstrated that KOR1 is primarily present in plasma membrane fractions but also at low levels in a tonoplast-enriched fraction (Nicol et al., 1998). Similarly, the KOR1 ortholog from tomato (Solanum lycopersicum) was found in the plasma membrane and fractions enriched for the Golgi apparatus (Brummell et al., 1997). A GFP-KOR1 fusion protein expressed with the Cauliflower mosaic virus 35S promoter accumulated in the Golgi apparatus and post-Golgi compartments and the tonoplast (Robert et al., 2005). Surprisingly for an enzyme involved in cellulose synthesis, the protein could not be detected at the plasma membrane. Using this construct, it was also shown that KOR1 undergoes regulated intracellular cycling (Robert et al., 2005).Although numerous genetic studies indicate that KOR1 is required for cellulose synthesis in primary and secondary cell walls and during cell plate formation (Nicol et al., 1998; Peng et al., 2000; Zuo et al., 2000; Lane et al., 2001; Sato et al., 2001; Szyjanowicz et al., 2004), its precise role in the cellulose synthesis process remains unclear. It has been suggested that KOR1 might be a component of the CSC (Read and Bacic, 2002). However, until now there has been no experimental evidence for this in Arabidopsis (Arabidopsis thaliana), either with coprecipitation experiments or with localization studies (Szyjanowicz et al., 2004; Robert et al., 2005; Desprez et al., 2007). Numerous hypotheses have been proposed to explain the paradoxical role of KOR1 in cellulose synthesis (Robert et al., 2004). KOR1 might have a proofreading activity involved in hydrolyzing disordered amorphous cellulose to relieve stress generated during the assembly of glucan chains in cellulose microfibrils (Mølhøj et al., 2002). Alternatively, KOR1 may determine the length of individual cellulose chains, either during cellulose synthesis or once the microfibril has been incorporated in the wall. A third hypothesis is that KOR1 releases the cellulose microfibril from the CSC before the complex is internalized from the plasma membrane (Somerville, 2006). Studies in cotton (Gossypium hirsutum) fiber extracts identified sitosterol glucoside as a primer for the cellulose synthesis and suggested that KOR1 could be involved in their cleavage from the nascent glucan chain (Peng et al., 2002). However, this scenario is unlikely, since, at least for the bacterial CESA, which is homologous to plant CESAs, there is no evidence for the existence of lipid-linked precursors, as shown by the three-dimensional structure of an active complex (Morgan et al., 2013).In this study, we first confirmed previous observations (Paredez et al., 2008) that, in the leaky kor1-1 mutant, the velocity of the CSCs is reduced compared with that in a wild-type background but that, in addition, the mutation affects the ability of the cellulose synthesis inhibitor CGA325′615 (hereafter referred to as CGA) to induce the accumulation of GFP-CESA3 in a microtubule-associated compartment (MASC/small compartments carrying cellulose synthase complexes [SmaCCs]; Crowell et al., 2009; Gutierrez et al., 2009). This indicates that KOR1 plays a role both in the synthesis of cellulose and in the intracellular trafficking of the CSC. Using gel filtration approaches, we identified KOR1 in fractions of high molecular mass, suggesting that KOR1 is present in membranes as part of a protein complex. We next analyzed the dynamics of GFP-KOR1 expressed in the kor1-1 mutant background under the control of its endogenous promoter. GFP-KOR1 is found in discrete particles at the plasma membrane in the same cells as GFP-CESAs (Crowell et al., 2009). GFP-KOR1 plasma membrane particles migrate along linear trajectories with comparable velocities to those observed for GFP-CESAs. The organization of GFP-KOR1 at the plasma membrane also requires the presence of an intact microtubule array, suggesting that KOR1 and CESA trajectories in the plasma membrane are regulated in the same manner. GFP-KOR1 and mCherry-CESA1 partially colocalize in the plasma membrane, Golgi, and post-Golgi compartments. Finally, we provide evidence for direct interaction between KOR1 and primary cell wall CESA proteins using the membrane-based yeast (Saccharomyces cerevisiae NMY51) two-hybrid (MbYTH) system (Timmers et al., 2009) and bimolecular fluorescence complementation (BiFC). Our data support a new model in which KOR1 is an integral part of the CSC, where it plays a role not only in the synthesis of cellulose but also in the intracellular trafficking of the CSC.     

Structural basis of TRAPPIII‐mediated Rab1 activation

The GTPase Rab1 is a master regulator of the early secretory pathway and is critical for autophagy. Rab1 activation is controlled by its guanine nucleotide exchange factor, the multisubunit TRAPPIII complex. Here, we report the 3.7 Å cryo‐EM structure of the Saccharomyces cerevisiae TRAPPIII complex bound to its substrate Rab1/Ypt1. The structure reveals the binding site for the Rab1/Ypt1 hypervariable domain, leading to a model for how the complex interacts with membranes during the activation reaction. We determined that stable membrane binding by the TRAPPIII complex is required for robust activation of Rab1/Ypt1 in vitro and in vivo, and is mediated by a conserved amphipathic α‐helix within the regulatory Trs85 subunit. Our results show that the Trs85 subunit serves as a membrane anchor, via its amphipathic helix, for the entire TRAPPIII complex. These findings provide a structural understanding of Rab activation on organelle and vesicle membranes.     

Kinetics of water loss from cells at subzero centigrade temperatures

A mathematical model is developed for the calculation of the kinetics of water loss from cells at subzero centigrade temperatures. In this model it is assumed that the cell surface membrane is permeable to water only, the protoplasm is a nonideal solution, the cells are spherical, and during the cooling process the cell temperature is not uniform inside the cell. It is also assumed that because of water loss due to cooling process the cell volume and the cell surface area reduce and the reductions in surface area and volume of the cell are functions of the amount of water loss from the cell. Based on this model, and for different conditions, the fractions of supercooled intracellular water remaining in the cells at various temperatures are calculated.It is shown that for cooling cells at subzero centigrade temperatures. (1) the consideration of Clausius-Clapeyron equation for vapor pressures of water and ice, instead of the exact vapor pressure relations, may produce errors in the prediction of the amount of water loss from the cells at high cooling rates only, (2) the assumption of intact cells will produce considerable deviation in the prediction of water loss from the cells as compared to the more realistic assumption of shrinkable cells, (3) the nonideality of protoplasm solution is very effective on the prediction of the amount of water loss from the cells, and (4) the assumption of uniform-temperature cells during the cooling process may be erroneous only for cells with small fractions of water in their protoplasms.     

Spawning seasons of Rasbora tawarensis (Pisces: Cyprinidae) in Lake Laut Tawar,Aceh Province,Indonesia

Background  

Rasbora tawarensis is an endemic freshwater fish in Lake Laut Tawar, Aceh Province, Indonesia. Unfortunately, its status is regarded as critical endangered with populations decreasing in recent years. To date no information on the spawning activities of the fish are available. Therefore, this study provides a contribution to the knowledge on reproductive biology of R. tawarensis especially on spawning seasons as well as basic information for conservation of the species.