Identification of neuron-specific promoters in Ciona intestinalis

We isolated 5' flanking regions of four genes, Ci-Galphai1, Ci-arr, Ci-vAChTP, and Ci-vGAT, each of which is expressed in distinct sets of neurons in the central nervous system of the ascidian Ciona intestinalis, and we examined their function by introducing green fluorescent protein (GFP)-fusion constructs into Ciona embryos. The reporter gene driven by the 5' flanking region of Ci-Galphai1, Ci-arr, and Ci-vAChTP recapitulated the endogenous gene expression patterns, while that of Ci-vGAT can drive GFP expression in particular subsets of neurons expressing the endogenous gene. Deletion analysis revealed that the Ci-Galphai1 promoter consists of multiple regulatory modules controlling the expression in different types of cells. The GFP fluorescence enabled visualization of cell bodies and axons of different sets of neurons in ascidian larvae. These promoters can be a powerful tool for studying molecular mechanisms of neuronal development as well as neuron networks and functions in ascidians.     

Reduction of natural adenovirus tropism to mouse liver by fiber-shaft exchange in combination with both CAR- and alphav integrin-binding ablation

The primary receptor, the coxsackievirus and adenovirus receptor (CAR), and the secondary receptor, αv integrins, are the tropism determinants of adenovirus (Ad) type 5. Inhibition of the interaction of both the fiber with CAR and the penton base with the αv integrin appears to be crucial to the development of targeted Ad vectors, which specifically transduce a given cell population. In this study, we developed Ad vectors with ablation of both CAR and αv integrin binding by mutating the fiber knob and the RGD motif of the penton base. We also replaced the fiber shaft domain with that derived from Ad type 35. High transduction efficiency in the mouse liver was suppressed approximately 130- to 270-fold by intravenous administration of the double-mutant Ad vectors, which mutated two domains each of the fiber knob and shaft and the RGD motif of the penton base compared with those of conventional Ad vectors (type 5). Most significantly, the triple-mutant Ad vector containing the fiber knob with ablation of CAR binding ability, the fiber shaft of Ad type 35, and the penton base with a deletion of the RGD motif mediated a >30,000-fold lower level of mouse liver transduction than the conventional Ad vectors. This triple-mutant Ad vector also mediated reduced transduction in other organs (the spleen, kidney, heart, and lung). Viral DNA analysis showed that systemically delivered triple-mutant Ad vector was primarily taken up by liver nonparenchymal cells and that most viral DNAs were easily degraded, resulting in little gene expression in the liver. These results suggest that the fiber knob, fiber shaft, and RGD motif of the penton base each plays an important role in Ad vector-mediated transduction to the mouse liver and that the triple-mutant Ad vector exhibits little tropism to any organs and appears to be a fundamental vector for targeted Ad vectors.     

Evolutional study on acetylcholine expression

Acetylcholine (ACh) is a well-known neurotransmitter in the cholinergic nervous systems of vertebrates and insects; however, there is only indirect evidence for its presence in lower invertebrates, such as plants and fungi. We therefore investigated the expression of ACh in invertebrates (sea squirt, sea urchin, trepang, squid, abalone, nereis, sea anemone, coral and sponge), plants (arabidopsis, eggplant, bamboo shoot, cedar, hinoki, pine, podcarp, fern, horsetail and moss), fungi (yeast and mushroom) and bacteria by assaying ACh content and synthesis, focusing on the presence of two synthetic enzymes, choline acetyltransferase (ChAT) and carnitine acetyltransferase (CarAT). Using a specific radioimmunoassay, ACh was detected in all samples tested. The levels varied considerably, however, with the upper portion of bamboo shoots having the highest content (2.9 micromol/g). ACh synthesis was also detected in all samples tested; moreover, the activity in most samples from the animal kingdom, as well as bamboo shoots and the stem of the shiitake mushroom, were sensitive to both ChAT and CarAT inhibitors. Levels of ACh synthesis were lower in samples from other plants, fungi and bacteria and were insensitive to ChAT and CarAT inhibitors. These findings demonstrate the presence of ACh and ACh-synthesizing activity in evolutionally primitive life as well as in more complex multicellular organisms. In the context of the recent discovery of non-neuronal ACh in various mammalian species, these findings suggest that ACh been expressed in organisms from the beginning of life, functioning as a local mediator as well as a neurotransmitter.     

IgE-mediated activation of NK cells through Fc gamma RIII

NK cells express Fc gamma RIII (CD16), which is responsible for IgG-dependent cell cytotoxicity and for production of several cytokines and chemokines. Whereas Fc gamma RIII on NK cells is composed of both Fc gamma RIII alpha and FcR gamma chains, that on mast cells is distinct from NK cells and made of Fc gamma RIII alpha, FcR beta, and FcR gamma. Mast cells show degranulation and release several mediators, which cause anaphylactic responses upon cross-linking of Fc gamma RIII as well as Fc epsilon RI with aggregated IgE. In this paper, we examined whether IgE activates NK cells through Fc gamma RIII on their cell surface. We found that NK cells produce several cytokines and chemokines related to an allergic reaction upon IgE stimulation. Furthermore, NK cells exhibited cytotoxicity against IgE-coated target cells in an Fc gamma RIII-dependent manner. These effects of IgE through Fc gamma RIII were not observed in NK cells from FcR gamma-deficient mice lacking Fc gamma RIII expression. Collectively, these results demonstrate that NK cells can be activated with IgE through Fc gamma RIII and exhibit both cytokine/chemokine production and Ab-dependent cell cytotoxicity. These data imply that not only mast cells but also NK cells may contribute to IgE-mediated allergic responses.     

Alternative splicing and gene structure of the transforming growth factor beta-activated kinase 1

We have identified a fourth splice variant of the TGF beta-activated kinase (TAK1), called TAK1-d, and identified an error in the previously published TAK1-c sequence. Our data shows that the c and d variants encode proteins whose carboxyl ends differ markedly from those of variants a and b. Analysis of the human TAK1 gene sequence, located at 6q16.1-q16.3, shows that the coding sequence is organised in 17 exons. The four splice variants result from alternative splicing of exons 12 and 16, the reading frame of exon 17 being determined by the presence or absence of exon 16. Study of the relative levels of expression of the four splice variants showed significant variations between tissues. Our evidence suggests that the alternative splicing of the TAK1 mRNA may have important functional implications.     

Purification and characterization of dissimilatory nitrate reductase from a denitrifying halophilic archaeon, Haloarcula marismortui

Dissimilatory nitrate reductase was purified from a denitrifying halophilic archaeon, Haloarcula marismortui, to an electrophoretically homogeneous state. The purified enzyme was inferred to be a homotetramer composed of a 63 kDa polypeptide. The electron paramagnetic resonance spectrum of the purified enzyme revealed typical rhombic signals which were ascribed to Mo(V) in the Mo-molybdopterin complex. Like the bacterial membrane-bound (Nar-) enzyme, the purified enzyme supported the catalysis of chlorate. The enzyme was activated in extreme saline conditions and the values of k(cat) and K(m) toward nitrate were 145 s(-1) and 79 microM, respectively, in the presence of 2.0 M NaCl.     

Construction of a diabody (small recombinant bispecific antibody) using a refolding system

Diabodies are the recombinant bispecific antibodies (BsAbs), constructed from heterogeneous single-chain antibodies. Usually, diabodies have been prepared from bacterial periplasmic fraction using a co-expression vector (i.e. genes encoding two chains were tandemly located under the same promoter). Some diabodies, however, cannot be expressed as a soluble material owing to inclusion body formation, which limits the utilization of diabodies in various fields. Here we report an improved method for the construction of diabodies using a refolding system. As a model, a bispecific diabody binding to adenocarcinoma-associated antigen MUC1 and to CD3 on T cells was studied. One chain consisted of a VH specific for MUC1 linked to a VL specific for CD3 with a short polypeptide linker (GGGGS). The second was composed of a VL specific for MUC1 linked to a VH specific for CD3. The two hetero scFvs were independently obtained from intracellular insoluble fractions of Escherichia coli, purified, mixed stoichiometrically (at an equivalent molar ratio of 1:1) and refolded. The refolded two hetero scFv has a hetero-dimeric structure, with complete specificity for both target cells [i.e. MUC1 positive cells and CD3 positive lymphokine-activated killer cells with a T cell phenotype (T-LAK)]. Evaluation of the in vitro efficacy of T-LAK with the diabody by growth inhibition assay of cancer cells demonstrated maximum growth inhibition of cancer cells to reach approximately 98% at an effector:target ratio (E:T ratio) of 10, almost identical with that with anti-MUC1xanti-CD3 chemically synthesized BsAbs (c-BsAbs). This is the first report of the construction of a diabody using a refolding system.     

Effect of stress-like concentrations of cortisol on the feedback potency of oestradiol in orchidectomized sheep

The effect of stress-like concentrations of cortisol on oestradiol-induced change in LH secretion and GnRH receptor expression was evaluated in orchidectomized sheep (wethers). Twenty-four wethers were assigned at random to one of the four treatment groups in a 2x2 factorial design (n=6 wethers/group). Wethers received cortisol (90 microg/kg/h; groups 2 and 4) or a comparable volume of cortisol delivery vehicle (groups 1 and 3) by continuous infusion for 48 h. During the final 24 h of infusion, wethers received oestradiol (6 ng/kg/h; groups 3 and 4) or oestradiol delivery vehicle (groups 1 and 2). The pattern of LH secretion was assessed during a 3-h period of intensive blood collection beginning 21 h after initiation of oestradiol infusion. Although neither cortisol nor oestradiol alone affected (P>0.05) mean serum concentration of LH or LH pulse frequency, serum LH and the frequency of secretory episodes of LH were significantly reduced (P<0.05) in wethers receiving cortisol and oestradiol in combination. Anterior pituitary tissue was collected at the end of the infusion period. Oestradiol increased (P<0.05) tissue concentrations of GnRH receptor and GnRH receptor mRNA. Although cortisol alone did not affect (P>0.05) basal concentrations of receptor or receptor mRNA, the magnitude of oestradiol-induced increase in GnRH receptor and GnRH receptor mRNA was significantly reduced in wethers receiving cortisol and oestradiol concurrently. Conversely, steady-state concentrations of mRNA encoding the LHbeta and FSHbeta subunits were increased (P<0.05) in wethers receiving cortisol. These observations demonstrate that stress-like concentrations of cortisol act in concert with oestradiol to suppress LH secretion. In addition, cortisol blocks oestradiol-dependent increase in pituitary tissue concentrations of GnRH receptor and GnRH receptor mRNA.