大鼠原代肝细胞培养方法的建立

目的：探索混合胶原凝胶培养肝细胞的方法,观察培养鼠肝细胞的功能与形态特征,用于评价中药十八反的作用机理。方法：两步法分离鼠肝细胞,与鼠尾胶原溶液混合接种于培养板,观察培养鼠肝细胞的形态学特征和生化指标。采用RT-PCR技术。检测药物对P450亚酶CYP3A1表达的影响,进一步确证该体系的可靠性。结果：双层胶原培养体系可观察到典型的肝细胞形态特征,肝细胞功能检测显示肝细胞合成分泌的尿素、白蛋白,而乳酸脱氢酶漏出量较少。药物对P450亚酶CYP3A1表达的影响呈良好的剂量依赖性,同时双层胶原具有保持肝细胞活性的优点。可作为原代肝细胞培养的条件。结论：混合胶原凝胶培养能保留体内的细胞功能和活性,特别是保留药物代谢酶的活性。     

Reciprocal regulation of brain and muscle Arnt-like protein 1 and peroxisome proliferator-activated receptor alpha defines a novel positive feedback loop in the rodent liver circadian clock

Recent evidence has emerged that peroxisome proliferator-activated receptor alpha (PPARalpha), which is largely involved in lipid metabolism, can play an important role in connecting circadian biology and metabolism. In the present study, we investigated the mechanisms by which PPARalpha influences the pacemakers acting in the central clock located in the suprachiasmatic nucleus and in the peripheral oscillator of the liver. We demonstrate that PPARalpha plays a specific role in the peripheral circadian control because it is required to maintain the circadian rhythm of the master clock gene brain and muscle Arnt-like protein 1 (bmal1) in vivo. This regulation occurs via a direct binding of PPARalpha on a potential PPARalpha response element located in the bmal1 promoter. Reversely, BMAL1 is an upstream regulator of PPARalpha gene expression. We further demonstrate that fenofibrate induces circadian rhythm of clock gene expression in cell culture and up-regulates hepatic bmal1 in vivo. Together, these results provide evidence for an additional regulatory feedback loop involving BMAL1 and PPARalpha in peripheral clocks.     

Pterostilbene protects vascular endothelial cells against oxidized low-density lipoprotein-induced apoptosis in vitro and in vivo

Vascular endothelial cell (VEC) apoptosis is the main event occurring during the development of atherosclerosis. Pterostilbene (PT), a natural dimethylated analog of resveratrol, has been the subject of intense research in cancer and inflammation. However, the protective effects of PT against oxidized low-density lipoprotein (oxLDL)-induced apoptosis in VECs have not been clarified. We investigated the anti-apoptotic effects of PT in vitro and in vivo in mice. PT at 0.1–5 μM possessed antioxidant properties comparable to that of trolox in a cell-free system. Exposure of human umbilical vein VECs (HUVECs) to oxLDL (200 μg/ml) induced cell shrinkage, chromatin condensation, nuclear fragmentation, and cell apoptosis, but PT protected against such injuries. In addition, PT injection strongly decreased the number of TUNEL-positive cells in the endothelium of atherosclerotic plaque from apoE^−/− mice. OxLDL increased reactive oxygen species (ROS) levels, NF-κB activation, p53 accumulation, apoptotic protein levels and caspases-9 and -3 activities and decreased mitochondrial membrane potential (MMP) and cytochrome c release in HUVECs. These alterations were attenuated by pretreatment with PT. PT inhibited the expression of lectin-like oxLDL receptor-1 (LOX-1) expression in vitro and in vivo. Cotreatment with PT and siRNA of LOX-1 synergistically reduced oxLDL-induced apoptosis in HUVECs. Overexpression of LOX-1 attenuated the protection by PT and suppressed the effects of PT on oxLDL-induced oxidative stress. PT may protect HUVECs against oxLDL-induced apoptosis by downregulating LOX-1-mediated activation through a pathway involving oxidative stress, p53, mitochondria, cytochrome c and caspase protease. PT might be a potential natural anti-apoptotic agent for the treatment of atherosclerosis.     

SAR and molecular mechanism study of novel acylhydrazone compounds targeting HIV-1 CA

We synthesized a series of acylhydrazone compounds bearing naturally occurring amino acids’ side chains as HIV assembly inhibitors. Biological evaluation indicated that the compounds had anti-SIV and capsid assembly inhibitory activities. The structure–activity relationship (SAR) study showed that compounds bearing proper aromatic side chains had potential antiviral activities. The molecular modeling experiments revealed the molecular mechanism that they could bind to CA in the same manner as CAP-1 and occupy two more grooves.     

X-Linked thrombocytopenia causing mutations in WASP (L46P and A47D) impair T cell chemotaxis

Background

Mutation in the Wiskott-Aldrich syndrome Protein (WASP) causes Wiskott-Aldrich syndrome (WAS), X-linked thrombocytopenia (XLT) and X-linked congenital neutropenia (XLN). The majority of missense mutations causing WAS and XLT are found in the WH1 (WASP Homology) domain of WASP, known to mediate interaction with WIP (WASP Interacting Protein) and CIB1 (Calcium and Integrin Binding).

Results

We analyzed two WASP missense mutants (L46P and A47D) causing XLT for their effects on T cell chemotaxis. Both mutants, WASP_R^L46P and WASP_R^A47D (S1-WASP shRNA resistant) expressed well in Jurkat^WASP-KD T cells (WASP knockdown), however expression of these two mutants did not rescue the chemotaxis defect of Jurkat^WASP-KD T cells towards SDF-1α. In addition Jurkat^WASP-KD T cells expressing these two WASP mutants were found to be defective in T cell polarization when stimulated with SDF-1α. WASP exists in a closed conformation in the presence of WIP, however both the mutants (WASP_R^L46P and WASP_R^A47D) were found to be in an open conformation as determined in the bi-molecular complementation assay. WASP protein undergoes proteolysis upon phosphorylation and this turnover of WASP is critical for T cell migration. Both the WASP mutants were found to be stable and have reduced tyrosine phosphorylation after stimulation with SDF-1α.

Conclusion

Thus our data suggest that missense mutations WASP_R^L46P or WASP_R^A47D affect the activity of WASP in T cell chemotaxis probably by affecting the turnover of the protein.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-014-0091-1) contains supplementary material, which is available to authorized users.     

Macrophagelike vacuolated renal tubular cells in the urine of a male with osmotic nephrosis associated with intravenous immunoglobulin therapy. A case report

BACKGROUND: Osmotic nephrosis is a form of renal tubular injury that has been found in patients treated with intravenous immunoglobulin (IVIG). CASE: A 46-year-old male who had two courses of chemotherapy for acute myelogenous leukemia was found to have refractory thrombocytopenia. After IVIG (Sandoglobulin 12%, Novartis) administration (1 g/kg) for five consecutive days, the patient became oliguric and eventually anuric on the fifth dose. Hemodialysis was initiated, and urine production was noted on day 2 of hospitalization. Routine cytologic examination of fresh, voided urine showed numerous macrophagelike, bland epithelial cells with abundant, multivacuolated cytoplasm. Cytokeratin immunostain revealed positivity, thus confirming the epithelial origin of these cells. CONCLUSION: To our knowledge, this is the first such case reported in the English-language cytology literature. Awareness of a patient's clinical history may be helpful in avoiding an incorrect diagnosis. Urine cytology may be useful in obtaining an early diagnosis of osmotic nephrosis in patients receiving high-dose IVIG therapy that may eliminate the need for a renal biopsy.     

Affinity Laminated Chromatography Membrane Built‐in Electrodes for Suppressing Polysulfide Shuttling in Lithium–Sulfur Batteries

Lithium–sulfur (Li–S) batteries are promising candidates for energy storage, but suffer from capacity and cycling challenges caused by the serious shuttling effect of polysulfide (PS) ions. To address these issues, a sodium alginate (SA)‐derived affinity laminated chromatography membrane built‐in electrode is designed. This is the first attempt to utilize this type of membrane, which is widely used for the selective adsorption of proteins, in the battery field. An ordered multilayer structure throughout the electrode can easily be obtained, and the number of membrane layers can be also conveniently controlled by varying the cross‐linking time of SA. The PS shuttling effect is efficiently suppressed and the permeability of PSs is reduced by enveloping the carbon/sulfur powder in ultrathin laminated chromatography membranes. As a result, these designed electrodes deliver a superhigh initial capacity of 1492 mA h g^?1, with a capacity retention almost 20% higher than the contrast. This low‐cost and easily mass‐producible strategy inspired by affinity chromatography is expected to effectively solve the PS shuttling problem toward high‐loading and long‐lifetime Li–S batteries in practice.     

Direct Visualization of the Interfacial Degradation of Cathode Coatings in Solid State Batteries: A Combined Experimental and Computational Study

The interfacial instability between a thiophosphate solid electrolyte and oxide cathodes results in rapid capacity fade and has driven the need for cathode coatings. In this work, the stability, evolution, and performance of uncoated, Li₂ZrO₃‐coated, and Li₃B₁₁O₁₈‐coated LiNi_0.5Co_0.2Mn_0.3O₂ cathodes are compared using first‐principles computations and electron microscopy characterization. Li₃B₁₁O₁₈ is identified as a superior coating that exhibits excellent oxidation/chemical stability, leading to substantially improved performance over cells with Li₂ZrO₃‐coated or uncoated cathodes. The chemical and structural origin of the different performance is interpreted using different microscopy techniques which enable the direct observation of the phase decomposition of the Li₂ZrO₃ coating. It is observed that Li is already extracted from the Li₂ZrO₃ in the first charge, leading to the formation of ZrO₂ nanocrystallites with loss of protection of the cathode. After 50 cycles separated (Co, Ni)‐sulfides and Mn‐sulfides can be observed within the Li₂ZrO₃‐coated material. This work illustrates the severity of the interfacial reactions between a thiophosphate electrolyte and oxide cathode and shows the importance of using coating materials that are absolutely stable at high voltage.