Synergism between two distinct elements of the HTLV-I enhancer during activation by the trans-activator of HTLV-I

We have conducted functional studies of the enhancer elements of human T-cell leukemia virus type I (HTLV-I) using the human T-cell lines Jurkat and MOLT 4, which are negative for HTLV-I, and MT-2 and TL-Mor, which carry the proviral genome of HTLV-I. Two distinct elements have been implicated in function of the HTLV-I enhancer. One is the 21-base-pair (bp) core element that is responsible for trans-activation by the HTLV-I trans-activator p40tax and that has the ability to bind to cyclic-AMP responsive element binding factor (CREB)-like factor(s). The other is a region interposed between the 21-bp elements. In this study we demonstrate that a subfragment (C26) in the region between the 21-bp elements is involved in trans-activation by p40tax, possibly through binding to an NF-kappa B-like nuclear factor or factors. Formation of the protein-DNA complex with the C26 subfragment was positively affected by p40tax. The C26 element conferred partial responsiveness to p40tax when linked to one copy of the 21-bp element that, by itself, showed little activation in response to p40tax. However, the C26 element alone, even when repeated, could not be activated by p40tax, unlike other NF-kappa B-binding elements. In contrast, the C26 element itself was profoundly activated upon stimulation with 12-O-tetradecanoylphorbol-13-acetate. These findings therefore suggest that the HTLV-I enhancer contains multiple functional elements, including binding sites for at least CREB- and NF-kappa B-like factors, which synergistically cooperate in activation of the HTLV-I enhancer in response to p40tax. Our results also demonstrate that TPA-dependent activation of the HTLV-I enhancer may be mediated through the C26 element.     

Galectin-9 Is a High Affinity IgE-binding Lectin with Anti-allergic Effect by Blocking IgE-Antigen Complex Formation

Galectin (Gal)-9 was first described as an eosinophil chemoattractant. With the progress in research, Gal-9 has come to be known as a versatile immunomodulator that is involved in various aspects of immune regulations, and the entire picture of the function still remains elusive. To uncover as-yet unknown activity of Gal-9, we have been examining the effect of the protein in various disease animal models. Here we show that Gal-9 attenuated asthmatic reaction in guinea pigs and suppressed passive-cutaneous anaphylaxis in mice. These results indicate the mast cell stabilizing effect of Gal-9. In vitro studies of mast cell degranulation involving RBL-2H3 cells demonstrated that Gal-9 suppressed degranulation from the cells stimulated by IgE plus antigen and that the inhibitory effect was completely abrogated in the presence of lactose, indicating lectin activity of Gal-9 is critical. We found that Gal-9 strongly and specifically bound IgE, which is a heavily glycosylated immunoglobulin, and that the interaction prevented IgE-antigen complex formation, clarifying the mode of action of the anti-degranulation effect. Gal-9 is expressed by several mast cells including mouse mast cell line MC/9. The fact that immunological stimuli of MC/9 cells augmented Gal-9 secretion from the cells implies that Gal-9 is an autocrine regulator of mast cell function to suppress excessive degranulation. Collectively, these findings shed light on a novel function of Gal-9 in mast cells and suggest a beneficial utility of Gal-9 for the treatment of allergic disorders including asthma.     

Free radicals in the 1900's: from in vitro to in vivo

Remarkable progress has been achieved in the past 100 years in the field of free radical chemistry, biology and medicine since the discovery of free radicals in 1900. Free radical-mediated processes play a major role in the present industrial chemistry, but they also cause deleterious effects on rubber, plastics, oil products and foods. The importance of free radicals in vivo has been recognized increasingly from both positive and negative sides. Free radicals play an important role in phagocytosis, the production of some biologically essential compounds and possibly cell signaling. At the same time, they may cause oxidative modification of biological molecules, which leads to oxidative damage and eventually to various diseases, cancer and aging. The role and beneficial effects of antioxidants against such oxidative stress support this view. Furthermore, novel issues have been continuously found in this fascinating and yet controversial field of free radicals in biology. In this short article, the past work, present problems and future perspectives of free radicals in life science will be briefly discussed.     

α-Tocopheryl phosphate: uptake, hydrolysis, and antioxidant action in cultured cells and mouse

α-Tocopheryl phosphate (α-TP), a water-soluble analogue of α-tocopherol, is found in humans, animals, and plants. α-TP is resistant to both acid and alkaline hydrolysis and may exert its own function in this form in vivo. In this study, the uptake, hydrolysis, and antioxidant action of α-TP were measured using α-TP with a deuterated methyl group, CD(3), at position 5 of the chroman ring (α-TP(CD3)). The hydrolysis of α-TP(CD3) was followed by measuring α-tocopherol containing the CD(3) group, α-T(CD3), in comparison to unlabeled α-tocopherol, α-T(CH3). α-TP(CD3) was incubated with cultured cells, and the intracellular α-T(CD3) formed was measured with HPLC-ECD and GC-MS. α-TP(CD3) was also administered to mice for 4 weeks by mixing in the diet, and α-T(CD3) was measured in plasma, liver, brain, heart, and testis to compare with endogenous unlabeled α-T(CH3). It was found that α-TP(CD3) was taken in and hydrolyzed readily to α-T(CD3) in cultured cells and in mice. The hydrolysis of α-TP(CD3) in cell culture medium was not observed. α-TP protected primary cortical neuronal cells from glutamate-induced cytotoxicity, and α-TP given to mice reduced the levels of lipid peroxidation products in plasma and liver. These results suggest that α-TP is readily hydrolyzed in vivo to α-T, which acts as an antioxidant, and that α-TP may be used as a water-soluble α-T precursor in intravenous fluids, in eye drops, or as a dietary supplement.     

Lipid phenotype of two distinct subpopulations of Mycobacterium bovis Bacillus Calmette-Guerin Tokyo 172 substrain

Bacillus Calmette-Guérin (BCG) Tokyo 172 is a predominant World Health Organization Reference Reagent for the BCG vaccine. Recently, the BCG Tokyo 172 substrain was reported to consist of two subpopulations with different colony morphologies, smooth and rough. Smooth colonies had a characteristic 22-bp deletion in Rv3405c of the region of difference (RD) 16 (type I), and rough colonies were complete in this region (type II). We hypothesized that the morphological difference is related to lipid phenotype and affects their antigenicity. We determined the lipid compositions and biosynthesis of types I and II. Scanning electron microscopy showed that type I was 1.5 times longer than type II. Phenolic glycolipid (PGL) and phthiocerol dimycocerosate (PDIM) were found only in type I. Although it has been reported that the RD16 is involved in the expression of PGL, type II did not possess PGL/PDIM. We examined the ppsA-E gene responsible for PGL/PDIM biosynthesis and found that the existence of PGL/PDIM in types I and II is caused by a ppsA gene mutation not regulated by the RD16. PGL suppressed the host recognition of total lipids via Toll-like receptor 2, and this suggests that PGL is antigenic and involved in host responses, acting as a cell wall component. This is the first report to show the difference between lipid phenotypes of types I and II. It is important to clarify the heterogeneity of BCG vaccine substrains to discuss and evaluate the quality, safety, and efficacy of the BCG vaccine.     

Cloning, sequencing, and characterization of multicopy suppressors of a mukB mutation in Escherichia coli

The mukB gene codes for a 177kDa protein, which might be a candidate for a force-generating enzyme in chromosome positioning in Escherichia coli. The mukB106 mutant produces normal-sized, anucleate cells and shows a temperature-sensitive colony formation. To Identify proteins interacting with the MukB protein, we isolated three multicopy suppressors (msmA, msmB, and msmC) to the temperature-sensitive colony formation of the mukB106 mutation. The msmA gene, which could not suppress the production of anucleate cells, was found to be identical to the dksA gene. The msmB and msmC genes suppressed the production of anucleate cells as well as the temperature-sensitive colony formation. However, none of them couid suppress both phenotypes in a mukB null mutation. DNA sequencing revealed that the msmB gene was identicai to the cspC gene and that the msmC gene had not been described before. A homology search revealed that the amino acid sequences of both MsmB and MsmC possessed high similarity to proteins containing the cold-shock domain, such as CspA of E. coliand the Y-box binding proteins of eukaryotes; this suggests that MsmB and MsmC might be DNA-binding proteins that recognize the CCAAT sequence. Hence, the msmB and msmC genes were renamed cspC and cspE, respectively. Possible mechanisms for suppression of the mukB106 mutation are discussed.     

The DNA Damage Checkpoint Regulates a Transition between Yeast and Hyphal Growth in Schizosaccharomyces japonicus

Dimorphic yeasts change between unicellular growth and filamentous growth. Many dimorphic yeasts species are pathogenic for humans and plants, being infectious as invasive hypha. We have studied the determinants of the dimorphic switch in the nonpathogenic fission yeast Schizosaccharomyces japonicus, which is evolutionarily close to the well-characterized fission yeast S. pombe. We report that camptothecin, an inhibitor of topoisomerase I, reversibly induced the unicellular to hyphal transition in S. japonicus at low concentrations of camptothecin that did not induce checkpoint arrest and the transition required the DNA checkpoint kinase Chk1. Furthermore, a mutation of chk1 induced hyphal transition without camptothecin. Thus, we identify a second function for Chk1 distinct from its role in checkpoint arrest. Activation of the switch from single cell bipolar growth to monopolar filamentous growth may assist cells to evade the source of DNA damage.Yeasts and molds are major members of the kingdom Fungi. Molds grow as multicellular filamentous hyphae. On the other hand, yeasts propagate in a unicellular fashion by budding or by binary fission. However, many types of yeast can switch their growth modes, changing from unicellular growth to filamentous branching multicellular hyphae. This hyphal transition can be induced by a wide variety of environmental changes ranging from pH to the nature of the carbon source, and many species of dimorphic yeasts that are pathogenic for humans and plants are infectious in the hyphal form (15, 20).Hyphal transition is a simple mode of cellular differentiation program that is turned on upon environmental changes. The fungi may differentiate to adapt to the environmental challenges. Especially in the case of Candida albicans strains that infect humans, the hyphal transition may function as an action to resist against attack from macrophages or neutrophils. Hyphae are more difficult to phagocytose (16). It can also eventually kill macrophages if hyphal transition is triggered after ingestion by macrophage (14). Indeed, C. albicans cells that cannot form hyphae are avirulent. However, inducing hyphal growth in pathogenic yeasts is not always readily achievable in the laboratory, and genetic analysis of the hyphal growth phase and transition to this phase is often limited by the lack of appropriate tools. Thus, genetically tractable nonpathogenic dimorphic yeasts are attractive models for investigating invasive hypha.The nonpathogenic fission yeast Schizosaccharomyces japonicus is evolutionarily close to the well-characterized fission yeast Schizosaccharomyces pombe (5, 24). S. japonicus is dimorphic, transiting between unicellular and hyphal growth, and thus offers itself as an appropriate model to study this differentiation mechanism and the requirements of hyphal growth (25). In S. japonicus, hyphal growth occurs naturally on most solid medium and can occur over a range of nutrient conditions (26). It has been proposed that a gradient of nitrogen in the substrate is necessary to both initiate and direct hyphal growth in S. japonicus (26). In this report we establish conditions to induce hyphal growth in a microchamber in liquid media. In addition, we show that a low dose of the topoisomerase inhibitor camptothecin (CPT) induces hyphal differentiation under rich nutrient conditions and identify a role for the DNA damage checkpoint response in promoting the CPT-dependent transition from unicellular to hyphal growth. Genetic analysis demonstrates that this role of the checkpoint is distinct from checkpoint arrest, and we suggest it may provide an opportunity for S. japonicus to grow away from sources of genotoxic stress.