Induction of antibody responses in mice immunized intranasally with Type I interferon as adjuvant and synergistic effect of chitosan

Type I IFNs are a range of host-derived molecules with adjuvant potential; they have been used for many years in the treatment of cancer and viral hepatitis. Therefore, the safety of IFNs for human use has been established. In this study, we evaluated the mucosal adjuvanticity of IFN-β administered intranasally to mice with diphtheria toxoid, and suggested a method to improve its adjuvanticity. When IFN-β alone was used as a mucosal adjuvant, no clear results were obtained. However, simultaneous administration of IFN-β and chitosan resulted in an enhancement of the specific serum immunoglobulin G (IgG) and IgA antibody responses, the mucosal IgA antibody response, and antitoxin titers. Furthermore, the intranasal administration of IFN-α alone resulted in a greater increase in antibody titer than IFN-β, and a synergistic effect with chitosan was also observed. These findings suggest that intranasal administration of chitosan and Type I IFNs may display an effective synergistic mucosal adjuvant activity.     

Growth trajectories of prenatal embryos of the deep-sea shark Chlamydoselachus anguineus (Chondrichthyes)

Chlamydoselachus anguineus, Garman 1884, commonly called the frilled shark, is a deep-sea shark species occurring up to depths of 1300 m. It is assumed to represent an ancient morphotype of sharks (e.g., terminal mouth opening, more than five gill slits) and thus is often considered to represent plesiomorphic traits for sharks. Therefore, its early ontogenetic developmental traits are important for understanding the evolution of its particular phenotype. Here, we established six stages for prenatal embryos and used linear measurements and geometric morphometrics to analyse changes in shape and size as well as their timing during different embryonic stages. Our results show a change in head shape and a relocation of the mouth opening at a late stage of development. We also detected a negative allometric growth of the head and especially the eye compared to the rest of the body and a sexual dimorphism in total body length, which differs from the known data for adults. A multivariate analysis of covariance shows a significant interaction of shape related to the logarithm of centroid size and developmental stage. Geometric morphometrics results indicate that the head shape changes as a covariate of body size while not accounting for differences between sexes. The growth pattern of stages 32 and 33 indicates a shift in head shape, thus highlighting the moment in development when the jaws start to elongate anteriorly to finally achieve the adult condition of terminal mouth opening rather than retaining the early embryonic subterminal position as is typical for sharks. Thus, the antero-terminal mouth opening of the frilled shark has to be considered a derived feature.     

Response of Merkel cells in the palatal rugae to the continuous mechanical stimulation by palatal plate

The aim of the present study was to investigate the responses of Merkel cells that are numerous in the palatine rugae, due to the continuous mechanical stimulation exerted by the palatal plate. Forty golden hamsters were used in this experiment. The palatal plate was made of adhesive resin and it was set on the palate of the animal. To exert a continuous pressure, a 0.8?mm elevation on the internal surface of the palatal plate was created at the middle portion of the fourth palatine ruga. Thereafter, the number of Merkel cells in the mucosa was calculated by immunohistochemical observation. Morphological changes of Merkel cells were examined by electron microscopy. There was significant difference among the control and any of the treated groups on the number of CK20 positive Merkel cells (p?<?0.05) and that numbers were decreased at the sites where continuous mechanical stimulation was exerted. Degeneration of the cytoplasm mitochondria and nerve endings, and a decrease in both the number of neurosecretory granules and cytoplasmic processes were observed. Furthermore, the presence of nuclear chromatin aggregation and fragmentation was recognized. The continuous mechanical stimulation by the palatal plate affected the responses of Merkel cells and nerve endings, thus inducing a decrease in the number of Merkel cells. A portion of these changes was also associated with the expression of apoptosis.     

Facilitated Hyperpolarization Signaling in Vascular Smooth Muscle-overexpressing TRIC-A Channels

The TRIC channel subtypes, namely TRIC-A and TRIC-B, are intracellular monovalent cation-specific channels and likely mediate counterion movements to support efficient Ca²⁺ release from the sarco/endoplasmic reticulum. Vascular smooth muscle cells (VSMCs) contain both TRIC subtypes and two Ca²⁺ release mechanisms; incidental opening of ryanodine receptors (RyRs) generates local Ca²⁺ sparks to induce hyperpolarization and relaxation, whereas agonist-induced activation of inositol trisphosphate receptors produces global Ca²⁺ transients causing contraction. Tric-a knock-out mice develop hypertension due to insufficient RyR-mediated Ca²⁺ sparks in VSMCs. Here we describe transgenic mice overexpressing TRIC-A channels under the control of a smooth muscle cell-specific promoter. The transgenic mice developed congenital hypotension. In Tric-a-overexpressing VSMCs from the transgenic mice, the resting membrane potential decreased because RyR-mediated Ca²⁺ sparks were facilitated and cell surface Ca²⁺-dependent K⁺ channels were hyperactivated. Under such hyperpolarized conditions, L-type Ca²⁺ channels were inactivated, and thus, the resting intracellular Ca²⁺ levels were reduced in Tric-a-overexpressing VSMCs. Moreover, Tric-a overexpression impaired inositol trisphosphate-sensitive stores to diminish agonist-induced Ca²⁺ signaling in VSMCs. These altered features likely reduced vascular tonus leading to the hypotensive phenotype. Our Tric-a-transgenic mice together with Tric-a knock-out mice indicate that TRIC-A channel density in VSMCs is responsible for controlling basal blood pressure at the whole-animal level.     

The role of the helper lipid dioleoylphosphatidylethanolamine (DOPE) for DNA transfection cooperating with a cationic lipid bearing ethylenediamine

Gene therapy is expected to treat various incurable diseases including viral infections, autoimmune disorders, and cancers. Cationic lipids (CL) have been used as carriers of therapeutic DNAs for gene therapy because they can form a complex with DNA and such a complex can be incorporated into cells and transport the bound DNA to cytosol. The CL/DNA complexes are called lipoplexes and categorized as a non-viral vector. Lipoplexes are often prepared by adding a neutral phospholipid dioleoylphosphatidylethanolamine (DOPE) to CL in order to enhance transfection. However, the role of DOPE is not fully understood. We synthesized a new CL having an ethylenediamine cationic head group, denoted by DA, and found that addition of DOPE to DA achieved a good efficiency, almost in the similar level of commonly used transfection reagent Lipofectamine 2000 (Invitrogen). The composition of DA:DOPE = 1:1 showed the highest efficiency. This lipoplex showed structural transition when pH was changed from 7 to 4, corresponding pH lowering in late endosome, while DOPE itself showed structural transition at more basic pH around 8. The present data showed that the DOPE/DA composition determines the structural transition pH and choosing a suitable pH, i.e., a suitable composition, is essential to increase the transfection efficiency.     

Colorimetric/fluorogenic detection of thiols by N-fused porphyrin in water

A water-soluble derivative of N-fused porphyrin (NFP) possessing four cationic side-arms (pPyNFP) serves as a unique class of colorimetric/fluorogenic reporters that selectively react with biothiols in aquaous media to afford N-confused porphyrin (NCP) derivatives, while other nucleophilic amino acids were inert under a wide range of pH conditions. Owing to the large difference of the optical properties between NCP and NFP, the transformation enabled selective detection of biothiols in colorimetric/fluorogenic manner, especially in the near-infrared region. To the best our knowledge, this is the first example of porphyrin-based thiol detection systems that use the direct attack of thiol group on the optical reporter.     

Isolation and Chemical Characterization of a Mating Pheromone Produced by Saccharomyces kluyveri

The pullulanase gene (pul) of Klebsiella aerogenes was transferred in vivo to Escherichia coli by using RP4:: Mu cts. The pul gene was expressed in E. coli, although the level of pullulanase activity in E. coli was lower than that in K. aerogenes, and the Pul⁺ transconjugants were relatively unstable in an unselective medium. Production of pullulanase, which is used to make maltose from starch, was induced in E. coli by pullulan, waxy maize amylopectin, soluble starch and maltose. When the transconjugant cells of E. coli were grown with pullulan or maltose, most pullulanase was produced intracellularly, whereas K. aerogenes produced pullulanase extracellularly. Retransfer of the pul_k gene from E. coli to K. aerogenes by conjugation resulted in an increase of the production of extracellular pullulanase.     

Structures of Ezomycins A1 and A2

The structures of ezomycins A₁. and A₂, antifungal antibiotics produced by a strain of Streptomyces, were determined as 1 and 2, respectively, by degradative and spectrometric studies.     

Isolation and Identification of a Sexual Hormone in Yeast

5-Fluorotryptophan (5FT), indolmycin (IM), 4-fluorotryptophan and 7-azatryptophan were found on screening to be tryptophan antagonists among various chemically synthesized and naturally occurring tryptophan analogues for the isolation of l-tryptophan (l-Trp) producing mutants of Bacillus subtilis K.

From among 5FT resistant mutants, potent l-Trp producers were obtained using an improved isolation medium. Growth of the isolated 5FT-resistant l-Trp producer, AJ 11709, was inhibited by IM. From among 5FT and IM resistant mutants, the best strain, AJ 11979, which produced 9.0 g/liter of l-Trp from 13% glucose on 120hr cultivation, was selected.