Purification and structural properties of gelsolin, a Ca2+-activated regulatory protein of macrophages

We describe the purification procedure and some of the physiochemical properties of gelsolin, a major Ca2+-dependent regulatory protein of actin gel-sol transformation in rabbit lung macrophages. Gelsolin accounts for the majority of Ca2+ control of actin gelation in macrophage extracts. It is a single polypeptide chain with an average molecular weight of 91,000 a Stokes radius of 44 A, a sedimentation coefficient (s20(0),w) of 4.9 S, an isoelectric point of 6.1, and a frictional ratio of 1.43. Gelsolin binds 2 mol of Ca2+ with high affinity (Ka 1.09 X 10(6) M-1) in the presence of 0.1 M KCl and 2 mM MgCl2.     

Reconstituted low density lipoprotein: A vehicle for the delivery of hydrophobic fluorescent probes to cells

Previous studies have shown that the cholesteryl ester core of plasma low density lipoprotein (LDL) can be extracted with heptane and replaced with a variety of hydrophobic molecules. In the present report we use this reconstitution technique to incorporate two fluorescent probes, 3-pyrenemethyl-23, 24-dinor-5-cholen-22-oate-3β-yl oleate (PMCA oleate) and dioleyl fluorescein, into heptane-extracted LDL. Both fluorescent lipoprotein preparations were shown to be useful probes for visualizing the receptor-mediated endocytosis of LDL in cultured human fibroblasts. When normal fibroblasts were incubated at 37°C with either of the fluorescent LDL preparations, fluorescent granules accumulated in the perinuclear region of the cell. In contrast, fibroblasts from patients with the homozygous form of familial hypercholesterolemia (FH) that lack functional LDL receptors did not accumulate visible fluorescent granules when incubated with the fluorescent reconstituted LDL. A fluorescence-activated cell sorter was used to quantify the fluorescence intensity of individual cells that had been incubated with LDL reconstituted with dioleyl fluorescein. With this technique a population of normal fibroblasts could be distinguished from a population of FH fibroblasts. The current studies demonstrate the feasibility of using fluorescent reconstituted LDL in conjunction with the cell sorter to isolate mutant cells lacking functional LDL receptors.     

Effects of cytochalasin B on low-density lipoproteins metabolism by cultured human fibroblasts.

The role of cytoplasmic microfilaments in the metabolism of low-density lipoprotein by human fibroblasts was studied with the aid of cytochalasin B. At concentrations of 5--40 nmol/ml cytochalasin increased the surface binding but decreased the endocytosis of 125I-labelled low-density lipoprotein. Subsequent studies indicated that these changes reflected a reduction of the rate of internalisation of low-density lipoprotein receptors. Independent inhibitory effects were also observed on low-density lipoprotein degradation and on the cellular release of the trichloroacetic acid-soluble degradation products.     

The elucidation of the effect of ammonium chloride on pyruvate distribution and pyruvate dehydrogenase interconversion in isolated rat hepatocytes

The distribution of pyruvate between cell compartments measured in isolated hepatocytes in the presence of lactate was in agreement with delta pH across plasma and mitochondrial membranes. In isolated liver mitochondria NH4Cl decreased the transmembrane potential (delta psi) by about 14 mV, whereas no change of delta pH was observed. In the presence of lactate or alanine NH4Cl increased the mitochondrial pyruvate concentration presumably due to the inhibition of the flux through pyruvate carboxylase. In the presence of lactate or alanine changes in the amount of the active form of pyruvate dehydrogenase (PDHa) were correlated with the mitochondrial pyruvate concentration, NH4Cl increased the amount of PDHa by lowering the mitochondrial ATP/ADP and NADH/NAD+ ratios.     

The HIV epidemic in China： history, response, and challenge

The first case of AIDS was reported in 1985 in China, but by the early 21st century, the government estimated that there were 840,000 citizens living with HIV/AIDS. The number is increasing rapidly. The major risk groups are injection drug users （IDUSs; 43%） and former plasma donors （27%）, but rates among heterosexual groups are rising rapidly. Sentinel surveillance was initiated in 1986, and now includes IDUs, men-who-have-sex-with-men, sexually transmitted disease clinic attendees, antenatal women, long-distance truck drivers, and sex workers. Although the government was slow to respond to the epidemic in the late 20th century, it has made a vigorous response in the early 21st century. Components of that response include implementation and evaluation of harm reduction programs for IDUs, education to increase knowledge and reduce stigma, treatment and social support for rural and poor HIV/AIDS patients, widespread testing, and increased funding for HIV/AIDS programs. International agencies have been generous in their support of the government initiatives. To successfully combat the epidemic, China needs to develop and train the necessary infrastructure to implement its intervention programs, particularly in the rural areas, to vigorously combat stigma and discrimination, support research especially in the universities and research institutions other than the China Centers for Disease Control, develop a system for efficient exchange of research and program information, and update legislation to reflect the current situation.     

Circular RNA AFF4 modulates osteogenic differentiation in BM-MSCs by activating SMAD1/5 pathway through miR-135a-5p/FNDC5/Irisin axis

Bone marrow-derived mesenchymal stem cells (BM-MSCs), the common progenitor cells of adipocytes and osteoblasts, have been recognized as the key mediator during bone formation. Herein, our study aim to investigate molecular mechanisms underlying circular RNA (circRNA) AFF4 (circ_AFF4)-regulated BM-MSCs osteogenesis. BM-MSCs were characterized by FACS, ARS, and ALP staining. Expression patterns of circ_AFF4, miR-135a-5p, FNDC5/Irisin, SMAD1/5, and osteogenesis markers, including ALP, BMP4, RUNX2, Spp1, and Colla1 were detected by qRT-PCR, western blot, or immunofluorescence staining, respectively. Interactions between circ_AFF4 and miR-135a-5p, FNDC5, and miR-135a-5p were analyzed using web tools including TargetScan, miRanda, and miRDB, and further confirmed by luciferase reporter assay and RNA pull-down. Complex formation between Irisin and Integrin αV was verified by Co-immunoprecipitation. To further verify the functional role of circ_AFF4 in vivo during bone formation, we conducted animal experiments harboring circ_AFF4 knockdown, and born samples were evaluated by immunohistochemistry, hematoxylin and eosin, and Masson staining. Circ_AFF4 was upregulated upon osteogenic differentiation induction in BM-MSCs, and miR-135a-5p expression declined as differentiation proceeds. Circ_AFF4 knockdown significantly inhibited osteogenesis potential in BM-MSCs. Circ_AFF4 stimulated FNDC5/Irisin expression through complementary binding to its downstream target molecule miR-135a-5p. Irisin formed an intermolecular complex with Integrin αV and activated the SMAD1/5 pathway during osteogenic differentiation. Our work revealed that circ_AFF4, acting as a sponge of miR-135a-5p, triggers the promotion of FNDC5/Irisin via activating the SMAD1/5 pathway to induce osteogenic differentiation in BM-MSCs. These findings gained a deeper insight into the circRNA-miRNA regulatory system in the bone marrow microenvironment and may improve our understanding of bone formation-related diseases at physiological and pathological levels.Subject terms: Stem cells, Diseases     

Impacts of benzophenone-type UV filters on cladoceran Daphnia carinata

Limnology - The impacts of three commonly used benzophenone-type UV filters including benzophenone (BP), 2-hydroxy-4-methoxy-benzophenone (BP3), and 2-hydroxy-4-methoxy-benzophenone-5-sulfonicacid...     

Prediction of the Mechanism of Action of Fusaricidin on Bacillus subtilis

Long-term use of antibiotics has engendered a large number of resistant pathogens, which pose a serious threat to human health. Here, we investigated the mechanism of fusaricidin antibacterial activity toward Bacillus subtilis and characterized the pathways responsible for drug resistance. We found that σ^w, an extracytoplasmic function sigma factor, plays an important role in the resistance to fusaricidins during the initial 5 minutes of drug addition. Approximately 18 genes were induced more than 3-fold, of which 66.7% are known to be regulated by σ^w. Over the following 3 h, fusaricidins induced 194 genes more than three-fold, and most were associated with classes of antibiotic-responsive stimulons. Moreover, the fusaricidin treatment increased the catabolism of fatty and amino acids but strongly repressed glucose decomposition and gluconeogenesis. In summary, our data provide insight into the mechanism of fusaricidin activity, on which we based our suggested strategies for the development of novel antibiotic agents.