In vitro assay for responsiveness of lymphocytes to transfer factor by a new leukocyte migration inhibitory test.

Transfer factor (TF) causes nonimmune lymphocytes to produce leukocyte migration inhibitory factor (LMIF) in the presence of purified protein derivative (PPD). The activity of TF was measured by leukocyte migration inhibitory test (LMIT). The LMIT was a modification of the conventional agarose droplet method. To express the activity of LMIF quantitatively and simply, LMIF titer was introduced. The LMIF titer was obtained from the combination of two factors, LMIF dilution and cell migration diameter, and therefore this made the LMIT much more sensitive as compared to the conventional LMIT. The responsiveness of lymphocytes from acute lymphoblastic leukemia (ALL) and from cell-mediated immunodeficiency in children to TF was assayed by LMIT. In ALL, the lymphocyte responsiveness was poor in relapse but improved with remission. The responsiveness was remarkably well in 3 patients with cell-mediated immunodeficiency. This method appears useful for the in vitro evaluation of responsiveness of lymphocytes to TF.     

Development of artificial restriction DNA cutter composed of Ce(Iv)/EDTA and PNA

An artificial restriction enzyme, which we developed recently by combining Ce(IV)/EDTA and peptide nucleic acids, was used for PCR-free construction of a fusion protein. The fusion protein was successfully expressed in mammalian cells. This artificial DNA cutter can be also applied to site-selective scission of huge DNAs. Promising features of this novel tool were concretely evidenced.     

Responses of Trachelospermum asiaticum (Apocynaceae) seedlings to growth in a light intensity gradient

We recorded the growth of 24 seedlings of Trachelospermum asiaticum, a root climber, placed between a light source and a wall. Shoot length of seedlings planted at the brightest points nearly matched the distance the shoot tips moved toward the wall surface. In contrast, although the seedlings planted at the darkest points did elongate, the tips moved an average distance of only 0.4 cm. Creeping shoots of T. asiaticum planted in brighter environments exhibit negative phototropism, which encourages them to grow rapidly toward dark places, allowing them to reach supporting hosts faster than those that germinate close to the host.     

Xenopus death-domain-containing proteins FADD and RIP1 synergistically activate JNK and NF-kappaB

BACKGROUND INFORMATION: Death receptors (DRs) induce intracellular signalling upon engagement of their cognate ligands, leading to apoptosis, cell survival or pro-inflammatory responses. In mammals, DR signalling is mediated by the recruitment of several DD (death domain)-containing molecules, such as FADD (Fas-associated DD) and RIP1 (receptor-interacting protein 1). RESULTS: To elucidate the molecular mechanisms of intracellular DR signalling in Xenopus, we have isolated cDNAs encoding xFADD (Xenopus FADD), and xRIP1 and its short isoform xRIP1beta, which is produced by alternative splicing of the xRIP1 gene. These DD-containing proteins interacted with Xenopus DR members xDR-M1 and xDR-M2 through their DDs in co-transfected HEK-293T cells. Overexpression of xFADD activated not only xCaspase 8, but also AP-1 (activator protein 1), which reflects activation of JNK (c-Jun N-terminal kinase) and NF-kappaB (nuclear factor kappaB). A comparative analysis of xRIP1, a kinase-dead mutant of xRIP1 and xRIP1beta indicated that the kinase activity of xRIP1 was required for the activation of AP-1 and NF-kappaB. Interestingly, xFADD and xRIP1 interacted with each other via their DDs, and the expression of a mutant xRIP1 containing only the DD (xRIP1-DD) repressed the xFADD-induced activation of NF-kappaB and AP-1. xFADD and xRIP1 synergistically induced the activation of AP-1 and NF-kappaB, both of which were partially mediated by TRAF2 (tumour-necrosis-factor-receptor-associated factor 2) and TAK1 (transforming-growth-factor-beta-activated kinase 1). We also found that the activation pathways of NF-kappaB induced by xDR-M2 were inhibited by xRIP1-DD. CONCLUSIONS: Xenopus FADD, RIP1 and its splice variant RIP1beta have been characterized. Interaction of xFADD and xRIP1 induced synergistic activation of JNK and NF-kappaB. In addition, the NF-kappaB activation induced by xDR-M2 was partially mediated by xRIP1.     

Inhibition of Furin/Proprotein Convertase-catalyzed Surface and Intracellular Processing by Small Molecules

Furin is a ubiquitously expressed proprotein convertase (PC) that plays a vital role in numerous disease processes including cancer metastasis, bacterial toxin activation (e.g. anthrax and Pseudomonas), and viral propagation (e.g. avian influenza and human immunodeficiency virus). To identify small molecule inhibitors of furin and related processing enzymes, we performed high-throughput screens of chemical diversity libraries utilizing both enzyme-based and cell-based assays. The screens identified partially overlapping sets of compounds that were further characterized for affinity, mechanism, and efficacy in additional cellular processing assays. Dicoumarols were identified as a class of compounds that inhibited furin non-competitively and reversibly with K_i values in the micromolar range. These compounds inhibited furin/furin-like activity both at the cell surface (protecting against anthrax toxin) and in the secretory pathway (blocking processing of the metastasis factor membrane-type 1 matrix metalloproteinase/MT1-MMP) at concentrations close to K_i values. Compounds tested exhibited distinct patterns of inhibition of other furin-family PCs (rat PACE4, human PC5/6 and human PC7), showing that dicoumarol derivatives might be developed as either generic or selective inhibitors of the PCs. The extensive clinical use, high bioavailability and relatively low toxicity of dicoumarols suggests that the dicoumarol structure will be a good starting point for development of drug-like inhibitors of furin and other PCs that can act both intracellularly and at the cell surface.Furin, is a subtilisin-related serine protease and member of the proprotein convertase (PCs)4 family that functions within the secretory and endocytic pathways and at the cell surface, cleaving proproteins at clusters of basic residues, typically of the form RX(K/R)R↓ (for reviews see Refs. 1–3). The specificity of furin and its yeast homologue Kex2 correlate well with the three-dimensional structures of their catalytic domains (4, 5). Ubiquitously expressed, furin has numerous known or suspected physiological substrates that include growth factors, receptors, coagulation proteins, plasma proteins (e.g. pro-von Willebrand factor), extracellular matrix components, and protease precursors (e.g. matrix metalloproteases) (2). Although the homozygous furin knock-out mouse exhibits embryonic lethality (6), analysis of liver-specific ablation suggests functional overlap with other PCs, such as PACE4, PC5/6, and PC7, that are also widely expressed and act in the constitutive secretory pathway (7). Furin activity contributes to numerous chronic pathological conditions, including Alzheimer disease (8), other non-Alzheimer cerebral amyloidoses (9), osteoarthritis (10), atherosclerosis (11), and tumor progression and malignancy (12). Moreover, activation by host cells of bacterial toxins such as anthrax toxin, Pseudomonas exotoxin A, diphtheria toxin (13), Shiga toxin (14), and Bordetella dermonecrotic toxin (15), requires cleavage by furin or other PCs. Furin or furin-like cleavage of viral envelope glycoproteins is necessary for propagation of many lipid-enveloped viral pathogens including H5N1 avian influenza (16), human immunodeficiency virus-1 (17), ebola (18), measles (19), cytomegalovirus (20), and flaviviruses (21). Even non-enveloped viruses, such as human papillomavirus, can require furin-type processing for entry into the cyotsol after endocytosis (22).The multiple roles for furin in human pathophysiology have made it a target of interest for development of therapeutic agents. Numerous protein- and peptide-based furin inhibitors have been devised (23). For the most part, these are not drug-like and their use as pharmaceutical agents is hampered by large size, instability, toxicity, and/or low cell permeability. Recently, 2,5-dideoxystreptamine derivatives have shown promise (24), although these molecules have yet to be examined for inhibition of intracellular processing. Important pathophysiological roles exist for furin at the cell surface, such as in the processing of anthrax protective antigen. However, maturation of other bacterial toxins, viral envelope glycoproteins, and metalloprotease precursors such as membrane-type 1 matrix metalloproteinase (MT1-MMP), a matrix metalloprotease whose activity contributes directly to degradation of extracellular matrix components and is important for angiogenesis, tumor invasion, and metastasis (25), require processing by furin in the trans Golgi network and endosomal compartments (2, 26).Here we report identification of drug-like small molecule inhibitors through simultaneous high-throughput screening (HTS) of chemical diversity libraries with both enzyme-based and cell-based assays for furin and furin-like activities. A preliminary report of the cell-based assay has been published elsewhere (27). Combining the results of the enzymatic screen with the cellular screen allowed identification of small molecule lead compounds with the desired properties of high affinity, high cell permeability, and low toxicity. Dicoumarols, which have an extensive pharmacological history (28), were identified in this study as a family of compounds that inhibited furin reversibly and non-competitively, also inhibited rat PACE4 (rPACE4), human PC5/6 (hPC5/6), and hPC7 and blocked both extracellular maturation of anthrax protective antigen (PA) and intracellular processing of MT1-MMP and other substrates.     

Superoxide radical- and peroxynitrite-scavenging activity of anthocyanins; structure-activity relationship and their synergism

Antioxidant activities of 15 purified bilberry anthocyanins together with pelargonidin 3-O-beta-D-glucopyranoside and 4'-O-methyl delphinidin 3-O-beta-D-glucopyranoside (MDp 3-glc), the major metabolite of delphinidin 3-O-beta-D-glucopyranoside (Dp 3-glc), were evaluated in order to study the structure-antioxidant activity relationship and any synergism among them in the mixture. Both aglycone structure and the attached sugar moiety affected the O*2- and ONOO- -scavenging activities, although the effect of the attached sugar moiety was smaller than that of the aglycone structure. The potency of activity toward the superoxide radical was in the following order: delphinidin > petunidin > malvidin =approximately cyanidin>(+)-catechin > peonidin > pelargonidin. The activity toward ONOO- was: delphinidin > cyanidin =approximately petunidin > malvidin =approximately (+)-catechin > peonidin > pelargonidin. It was confirmed that methylation of 4'-OH markedly reduced the antioxidant activity of anthocyanin. Further, it was revealed that synergism occurred in both - and ONOO- -scavenging activities among the anthocyanins in the mixture.     

Early developmental stages of a protozoan parasite, Marteilioides chungmuensis (Paramyxea), the causative agent of the ovary enlargement disease in the Pacific oyster, Crassostrea gigas

A paramyxea, Marteilioides chungmuensis, causes the irregular enlargement of the ovary in the Pacific oyster, Crassostrea gigas in Korea and Japan. The knowledge about the life cycle of the parasite has been limited to the sporulation stages within the oocyte of oysters. In this study, we used the parasite-specific DNA probes and electron microscopy to experimentally infected oysters in a field and successfully clarified early developmental stages of the parasite. The parasite invaded the oysters through the epithelial tissues of the gills, mantle and labial palps. Extrasporogony repeatedly occurred in the connective tissues by binary fusion. The inner cell of the extrasporogonic stage migrated into the gonadal epithelium, invaded the oocyte to start sporulation.     

Furin inhibition by compounds of copper and zinc

Furin, a human subtilisin-related proprotein convertase (SPC), is emerging as an important pharmaceutical target because it processes vital proteins of many aggressive pathogens. Furin inhibitors reported as yet are peptide derivatives and proteins, with the exception of andrographolides, which are natural compounds. Here we report that the small and highly stable compounds M(chelate)Cl(2) (M is copper or zinc) inhibit furin and Kex2, with Cu(TTP)Cl(2) and Zn(TTP)Cl(2) as the most efficient inhibitors. (TTP is 4'-[p-tolyl]-2,2 ':6',2"-terpyridine.) Inhibition is irreversible, competitive with substrate, and affected by substituents on the chelate. The free chelates are not inhibitors. Solvated Zn(2+) is less potent than its complexes. This is true also for copper and Kex2. However, solvated Cu(2+) (k(on) of 25,000 +/- 2,500 s(-1)) is more potent than Cu(TTP)Cl(2) (k(on) = 140 +/- 13 s(-1) and allows recovery of furin activity prior to a second inhibition phase. A mechanism that involves coordination to the catalytic histidine is proposed for all inhibitors. Target specificity is indicated by the fact that these metal chelate inhibitors are much less potent toward Kex2, the yeast homologue of furin. For example, k(on) with Zn(TTP)Cl(2) is 120 +/- 20 s(-1) for furin, but only 1.2 +/- 0.1 s(-1) for Kex2.     

Predominant role of basicity of leaving group in alpha-effect for nucleophilic ester cleavage

It has been found that alpha-effects in nucleophilic reactions, unexpectedly large nucleophilicity due to adjacent unpaired electrons, are strongly dependent on the structure of substrate. The nucleophilic cleavages of 4-nitrobenzoate esters and 4-methylbenzoate esters by HOO- have been systematically investigated in detail. When the leaving groups of substrates are sufficiently good (aryl, 2,2,2-trifluoroethyl, and 2,2-dichloroethyl esters), alpha-effect is evident. However, this effect drastically decreases as the leaving group gets poorer, and is only marginal for the cleavages of 2-fluoroethyl and methyl esters. In the nucleophilic cleavages by salicylaldoxime and acetohydroxamic acid, alpha-effect is also notable only for the esters having good leaving groups. These enormous dependences of alpha-effects on the substrate-structure have been interpreted in terms of the difference in the position of transition-state in the reaction coordinate.