Cytolytic Activity Released from Tetrahymena1

Using a hemolytic assay system, we have detected cytolytic activities in extracellular medium from Tetrahymena thermophila and T. pyriformis. In addition, we have identified two phospholipase activities (types A and C) from the same source by thin layer chromatography analysis of the breakdown products of a phospholipid preparation. The hemolytic activity peaks at low pH values. It is inhibited by egg lecithin, supporting the view that a phospholipase activity is involved in the cytolytic processes. Cytolytic activities may play important roles in Tetrahymena, both in nutrition, especially in parasitic and scavenger forms, and in defense against predators. Tetrahymena is probably partly protected from its own released cytolytic phospholipase by having a high proportion of phosphonolipids on its surface membrane.     

JNK1 Phosphorylates SIRT1 and Promotes Its Enzymatic Activity

SIRT1 is a NAD-dependent deacetylase that regulates a variety of pathways including the stress protection pathway. SIRT1 deacetylates a number of protein substrates, including histones, FOXOs, PGC-1α, and p53, leading to cellular protection. We identified a functional interaction between cJUN N-terminal kinase (JNK1) and SIRT1 by coimmunoprecipitation of endogenous proteins. The interaction between JNK1 and SIRT1 was identified under conditions of oxidative stress and required activation of JNK1 via phosphorylation. Modulation of SIRT1 activity or protein levels using nicotinamide or RNAi did not modify JNK1 activity as measured by its ability to phosphorylate cJUN. In contrast, human SIRT1 was phosphorylated by JNK1 on three sites: Ser27, Ser47, and Thr530 and this phosphorylation of SIRT1 increased its nuclear localization and enzymatic activity. Surprisingly, JNK1 phosphorylation of SIRT1 showed substrate specificity resulting in deacetylation of histone H3, but not p53. These findings identify a mechanism for regulation of SIRT1 enzymatic activity in response to oxidative stress and shed new light on its role in the stress protection pathway.     

Ceramide 1-Phosphate Phosphatase Activity in Brain

Recent studies have implicated sphingolipids in a variety of intracellular signaling systems. The finding that a calcium-stimulated ceramide kinase copurifies with neurotransmitter-containing vesicles suggests that ceramide, or one of its metabolites, has a role in neurotransmitter release. As a step toward understanding the role of ceramide kinase in vesicle functioning, this study sought to determine the metabolic fate of the product, ceramide 1-phosphate. We report that ceramide 1-phosphate is not deacylated by brain ceramidases to produce sphingosine 1-phosphate. It is, however, the substrate for a phosphatase activity that we name ceramide 1-phosphate phosphatase (CPPase). Subcellular fractionation studies suggest that CPPase is found in the synaptic terminal and is associated with both synaptic vesicle and plasma membranes. Divalent cations, most notably calcium, inhibit CPPase activity although not at concentrations that activate ceramide kinase. The existence of both ceramide kinase and CPPase activities at the synapse suggests that ceramide 1-phosphate production regulates some aspect of synaptic vesicle functioning.     

In Vitro Activity of Coumermycin A1

The in vitro activity of coumermycin A(1) was compared with that of novobiocin, ampicillin, and minocycline. Coumermycin was found to be the most active antibiotic of the four against Staphylococcus aureus. It was about 50 times more active than novobiocin or minocycline against the strains tested. Coumermycin also showed good activity against group A streptococci and pneumococci, moderate activity against Escherichia coli, indole-positive Proteus species, and Pseudomonas aeruginosa, and poor activity against Klebsiella-Enterobacter and enterococci. Against P. mirabilis, however, coumermycin activity was almost equal to that of ampicillin. The new antibiotic was further found to be greatly reduced in activity in the presence of plasma, but its minimal inhibitory concentration was not greatly affected by inoculum size. Coumermycin was found to be bacteriostatic in its action, and resistance to it developed slowly. Also, cross-resistance was present with novobiocin but absent with ampicillin or minocycline.     

ArfGAP1 Activity and COPI Vesicle Biogenesis

Golgi-derived coat protein I (COPI) vesicles mediate transport in the early secretory pathway. The minimal machinery required for COPI vesicle formation from Golgi membranes in vitro consists of (i) the hetero-heptameric protein complex coatomer, (ii) the small guanosine triphosphatase ADP-ribosylation factor 1 (Arf1) and (iii) transmembrane proteins that function as coat receptors, such as p24 proteins. Various and opposing reports exist on a role of ArfGAP1 in COPI vesicle biogenesis. In this study, we show that, in contrast to data in the literature, ArfGAP1 is not required for COPI vesicle formation. To investigate roles of ArfGAP1 in vesicle formation, we titrated the enzyme into a defined reconstitution assay to form and purify COPI vesicles. We find that catalytic amounts of Arf1GAP1 significantly reduce the yield of purified COPI vesicles and that Arf1 rather than ArfGAP1 constitutes a stoichiometric component of the COPI coat. Combining the controversial reports with the results presented in this study, we suggest a novel role for ArfGAP1 in membrane trafficking.     

An Organofluorophosphate-Hydrolyzing Activity in Tetrahymena thermophila1

An enzymatic activity that hydrolyzes O,O-diisopropylphosphofluoridate (DFP) and O-1,2,2–trimethylpropylmethyl-phosphonofluoridate (Soman) was discovered in the ciliate protozoan Tetrahymena thermophila. The enzymatic activity classifies the protein as Mazur-type similar to that found in hog kidney and Escherichia coli. The rate of hydrolysis of Soman by the Tetrahymena-extract is the highest, on a per gram of extract basis, of any eucaryote. The molecular weight is approximately 75,400 as determined by Sephacryl column chromatography. A maximum fifteen-fold purification has been achieved. Potential exists for the detoxification and one-step detection of common organofluorophosphate pollutants. Additionally, Tetrahymena should prove an easier subject for manipulation than mammalian or squid sources. Protozoa may be a potentially important source of detoxification and degradation enzymes for other environmental contaminants.     

具有抑菌作用的放线菌GNF1的分离鉴定和生物学活性的研究

从几种复合微生物有机肥中分离出一系列不同的菌株,与实验室保存的菌株GNW(自生固氮菌)和HP2(解磷菌)混合接种培养,检测是否因基因杂交、突变等原因而产生具有抑菌作用的菌株.结果分离出一株具有抑菌作用的放线菌菌株GNF1,根据其形态学特征、生理生化特征和基于16S rRNA基因序列的系统发育分析结果,鉴定其属于链霉菌属(Streptomyces)的一个菌株,GNF1菌株的代谢产物中存在具有抑菌作用的活性成分,与植物根际促生菌GNW、HP2以及某些原核病原微生物共培养培养时能明显抑制它们的生长.     

萘查耳酮类化合物的设计合成及对CYP1B1酶的抑制活性评价

目的:设计并合成几类新型的萘查耳酮衍生物,初步测定其对CYP1B1酶的抑制活性,筛选具有良好抗癌作用的CYP1B1抑制剂。方法:以1,5-二羟基萘为原料,首先合成两个重要的中间体2-乙酰基-1,4,5,8-四甲氧基萘、1,5,6-三甲氧基-2-萘乙酮,然后利用羟醛缩合反应合成所需要的化合物。结果:合成了19个目标化合物,其结构均经过核磁共振氢谱确证,所有化合物均为全新化合物。对所合成的新化合物均进行了EROD酶实验测定。结论:所合成的化合物均具有较强的CYP1B1酶一抑制活性,其中4-1、4-2、5'-1对CYP1B1的抑制活性强于CYP1B1强抑制剂α-萘黄酮(IC50=11 nmol/L),他们的IC50分别为6.5、0.47、8nmol/L。     

The C-Terminal Sequence of IFITM1 Regulates Its Anti-HIV-1 Activity

The interferon-inducible transmembrane (IFITM) proteins inhibit a wide range of viruses. We previously reported the inhibition of human immunodeficiency virus type 1 (HIV-1) strain BH10 by human IFITM1, 2 and 3. It is unknown whether other HIV-1 strains are similarly inhibited by IFITMs and whether there exists viral countermeasure to overcome IFITM inhibition. We report here that the HIV-1 NL4-3 strain (HIV-1_NL4-3) is not restricted by IFITM1 and its viral envelope glycoprotein is partly responsible for this insensitivity. However, HIV-1_NL4-3 is profoundly inhibited by an IFITM1 mutant, known as Δ(117–125), which is deleted of 9 amino acids at the C-terminus. In contrast to the wild type IFITM1, which does not affect HIV-1 entry, the Δ(117–125) mutant diminishes HIV-1_NL4-3 entry by 3-fold. This inhibition correlates with the predominant localization of Δ(117–125) to the plasma membrane where HIV-1 entry occurs. In spite of strong conservation of IFITM1 among most species, mouse IFITM1 is 19 amino acids shorter at its C-terminus as compared to human IFITM1 and, like the human IFITM1 mutant Δ(117–125), mouse IFITM1 also inhibits HIV-1 entry. This is the first report illustrating the role of viral envelope protein in overcoming IFITM1 restriction. The results also demonstrate the importance of the C-terminal region of IFITM1 in modulating the antiviral function through controlling protein subcellular localization.     

Cysteine-independent Catalase-like Activity of Vertebrate Peroxiredoxin 1 (Prx1)

Peroxiredoxins (Prxs) are a ubiquitous family of antioxidant proteins that are known as thioredoxin peroxidases. Here we report that Prx1 proteins from Tetraodon nigroviridis and humans also possess a previously unknown catalase-like activity that is independent of Cys residues and reductants but dependent on iron. We identified that the GVL motif was essential to the catalase (CAT)-like activity of Prx1 but not to the Cys-dependent thioredoxin peroxidase (POX) activity, and we generated mutants lacking POX and/or CAT activities for individually delineating their functional features. We discovered that the TnPrx1 POX and CAT activities possessed different kinetic features in reducing H₂O₂. The overexpression of wild-type TnPrx1 and mutants differentially regulated the intracellular levels of reactive oxygen species and p38 phosphorylation in HEK-293T cells treated with H₂O₂. These observations suggest that the dual antioxidant activities of Prx1 may be crucial for organisms to mediate intracellular redox homeostasis.     

三七皂苷Rg1对tPA和PAI-1活性的调节作用

探讨三七皂苷Rg1对组织型纤溶酶原激活物(tPA)和纤溶酶原激活物抑制物(PAI-1)活性的调节作用。运用发色底物方法测定三七皂苷Rg1在体外和静脉注射对家兔血浆纤溶酶原激活物(tPA)和血浆或血小板释放的纤溶酶原激活物抑制物(PAI-1)水平的影响。结果表明,三七皂苷Rg1在体外呈浓度依赖性明显抑制血浆PAI-1活性,同时提高血浆tPA活性;30和60 mg/kg的三七皂苷Rg1静脉注射显著抑制血浆PAI-1活性,提高血浆tPA活性,同时降低凝血酶激活的血小板所释放的PAI-1水平。本实验提示三七皂苷Rg1能抑制PAI-1活性,同时升高tPA活性可能是其抗血栓作用的分子机制之一。     

Ethylene-Enhanced 1-Aminocyclopropane-1-carboxylic Acid Synthase Activity in Ripening Apples

Apples (Malus sylvestris Mill, cv Golden Delicious) were treated before harvest with aminoethoxyvinylglycine (AVG). AVG is presumed to reversibly inhibit 1-aminocyclopropane-1-carboxylic acid (ACC) activity, but not the formation of ACC synthase. AVG treatment effectively blocked initiation of autocatalytic ethylene production and ripening of harvested apples. Exogenous ethylene induced extractable ACC synthase activity and ripening in AVG-treated apples. Removal of exogenous ethylene caused a rapid decline in ACC synthase activity and in CO₂ production. The results with ripened, AVG-treated apples indicate (a) a dose-response relationship between ethylene and enhancement of ACC synthase activity with a half-maximal response at approximately 0.8 μl/l ethylene; (b) reversal of ethylene-enhanced ACC synthase activity by CO₂; (c) enhancement of ACC synthase activity by the ethylene-activity analog propylene.

Induction of ACC synthase activity, autocatalytic ethylene production, and ripening of preclimacteric apples not treated with AVG were delayed by 6 and 10% CO₂, but not by 1.25% CO₂. However, each of these CO₂ concentrations reduced the rate of increase of ACC synthase activity.

     

Blood Monocyte Chemotactic Protein-1 (MCP-1) and Adapted Disease Activity Score28-MCP-1: Favorable Indicators for Rheumatoid Arthritis Activity

Objective

We assessed blood pentraxin 3 (PTX3) and macrophage chemotactic factor-1 (MCP-1) levels as indicators of disease activity in rheumatoid arthritis (RA) patients, because data on disease activity score 28 (DAS28)-erythrocyte sedimentation rate (ESR) and DAS28-C-reactive protein (CRP) are still imperfect.

Methods

In 111 patients with RA, we examined longitudinal and cross-sectional correlations of blood PTX3, MCP-1, CRP, and ESR levels with measures of clinical arthritic activity, namely, swollen joint count (SJC), tender joint count (TJC), visual analog scale for general health (GH), DAS28, and adapted DAS28-MCP-1.

Results

Blood MCP-1, but not PTX3, was significantly correlated with SJC, TJC, DAS28, and DAS28-CRP. DAS28-MCP-1 was strongly correlated with DAS28 (r  = 0.984, P<0.001) and DAS28-CRP (r  = 0.971, P<0.001), and modestly correlated with CRP (r  = 0.350, P<0.001), and ESR (r  = 0.386, P<0.001). Similarly, the duration of arthritic symptoms, but not sex, was significantly correlated with variables of arthritic activity. In particular, DAS28-MCP-1 significantly correlated with DAS28 during a 6-month period (r  = 0.944, P<0.001; r  = 0.951, P<0.001; r  = 0.862, P<0.001; and r  = 0.865, P<0.001 for month 0, 1, 3, and 6, respectively).

Conclusion

Blood MCP-1 and adapted DAS28-MCP-1, but not blood PTX3, may be useful in monitoring RA activity.     

Cytolytic Activity of Naegleria fowleri Cell-free Extract1

ABSTRACT. The cytotoxic activity of a cell-free extract of Naegleria fowleri amebae on B103 rat nerve cells in culture was investigated. The cell-free extract was prepared by subjecting lysed amebae to centrifugation at 100,000 g for 1 h, precipitation of the supernatant fluid with 30–60% saturated ammonium sulfate, and desalting by group exclusion chromatography utilizing Sephadex G-25. The supernatant fluid recovered from this procedure was termed the soluble fraction. The Naegleria cytotoxic activity present in the soluble fraction was assayed by ⁵¹Cr released from labeled B103 cells. The Naegleria soluble fraction, when added to nerve cells, elicited blebs on the B103 target cell surface within 5 min after exposure to the fraction. Later, holes were observed in the B103 cell plasma membrane. These alterations were never observed on untreated B103 cells. Phospholipase A, phospholipase C, and protease activities were associated with the desalted ammonium sulfate-precipitable cytotoxic activity of N. fowleri cell-free lysate. The cytotoxic activity was impaired by ethylenediamine-tetraacetate (EDTA), phospholipase A inhibitor (Rosenthal's reagent), heating at 50°C for 15 min, or incubation at pH 10 for 60 min. Repeated freeze-thawing and inhibitors of proteolytic enzymes had no effect on the cytotoxic activity. Small amounts of ethanol (5% v/v) enhanced cytotoxic activity of the fraction. Phospholipases A and C, as well as other as yet unidentified cytolytic factors may be responsible for producing ⁵¹Cr release from target cells by the soluble fraction of N. fowleri extracts.     

Modulation of DMT1 Activity by Redox Compounds

Iron(II) exacerbates the effects of oxidative stress via the Fenton reaction. A number of human diseases are associated with iron accumulation including ischemia-reperfusion injury, inflammation and certain neurodegenerative diseases. The functional properties and localization in plasma membrane of cells and endosomes suggest an important role for the divalent metal transporter DMT1 (also known as DCT1 and Nramp2) in iron transport and cellular iron homeostasis. Although iron metabolism is strictly controlled and the activity of DMT1 is central in controlling iron homeostasis, no regulatory mechanisms for DMT1 have been so far identified. Our studies show that the activity of DMT1 is modulated by compounds that affect its redox status. We also show that both iron and zinc are transported by DMT1 when expressed in Xenopus laevis oocytes. Radiotracer uptake and electrophysiological measurements revealed that H₂O₂ and Hg²⁺ treatments result in substantial inhibition of DMT1. These findings may have a profound relevance from a physiological and pathophysiological standpoint. Present address for D.T.: Department of Neurology, Cecil B. Day Laboratory for Neuromuscular Research, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA