Structure-activity studies of heptapeptide derivatives related to substance P, neurokinin A, B and other tachykinins on smooth muscles

Diverse C-terminal heptapeptide derivatives related to substance P, kassinin, physalaemin, neurokinin A and B were synthesized and the contracting activities on the guinea pig ileum as well as rat duodenum were compared. In the partial sequence of C-terminal of tachykinin peptides, -I-II-Phe-III-Gly-Leu-Met-NH2, the combination of amino acid residues at positions I and III have significant roles in contraction of smooth muscle. For the activation of rat duodenal muscle (SP-E), Asp(I) and aliphatic amino acid(III), and for guinea pig ileal muscle(SP-P), Gln(I) and aromatic amino acid(III) are essential. Moreover, guinea pig ileum is sensitive to a full sequence of neurokinin peptides.     

Quantitative determination of sulfated glycopeptide by two-dimensional electrophoresis on cellulose acetate membrane

Quantitative determination of the sulfated glycoproteins present in tissue and secretion fluid was performed. After digestion of the specimen with pronase in order to convert glycoproteins to glycopeptides, the sulfated glycopeptides were separated from a mixture of acidic glycans (glycosaminoglycans, sialoglycopeptides and sulfated glycopeptides) by two-dimensional electrophoresis on cellulose acetate membrane [(1986) J. Biochem. Biophys. Methods 12, 239-246]. After staining with alcian blue, the spot of sulfated glycopeptide on the cellulose acetate membrane was cut out, and then only the dye bound to the sulfated glycopeptide was extracted with a 5% cetylpyridinium chloride solution at 100 degrees C for 15 min. The extract was then measured by absorbance at 615 nm using an authentic sulfated glycopeptide as a standard. This method facilitated the determination of sulfated glycopeptides, which were separated from other acidic glycans, within the range 0-25 micrograms.     

Structure of Novel Enzyme in Mannan Biodegradation Process 4-O-β-d-Mannosyl-d-Glucose Phosphorylase MGP

The crystal structure of a novel component of the mannan biodegradation system, 4-O-β-d-mannosyl-d-glucose phosphorylase (MGP), was determined to a 1.68-Å resolution. The structure of the enzyme revealed a unique homohexameric structure, which was formed by using two helices attached to the N-terminus and C-terminus as a tab for sticking between subunits. The structures of MGP complexes with genuine substrates, 4-O-β-d-mannosyl-d-glucose and phosphate, and the product d-mannose-1-phosphate were also determined. The complex structures revealed that the invariant residue Asp131, which is supposed to be the general acid/base, did not exist close to the glycosidic Glc-O4 atom, which should be protonated in the catalytic reaction. Also, no solvent molecule that might mediate a proton transfer from Asp131 was observed in the substrate complex structure, suggesting that the catalytic mechanism of MGP is different from those of known disaccharide phosphorylases.     

Transient and permanent gene transfer into the brain of the teleost fish medaka (Oryzias latipes) using human adenovirus and the Cre-loxP system

In this study, we demonstrated that human type-5 adenovirus infected the brain of the teleost fish, medaka (Oryzias latipes), in vivo. Injection of adenoviral vector into the mesencephalic ventricle of medaka larvae induced the expression of reporter genes in some parts of the telencephalon, the periventricular area of the mesencephalon and diencephalon, and the cerebellum. Additionally, the Cre-loxP system works in medaka brains using transgenic medaka carrying a vector containing DsRed2, flanked by loxP sites under control of the β-actin promoter and downstream promoterless enhanced green fluorescent protein (EGFP). We demonstrated that the presence of green fluorescence depended on injection of adenoviral vector expressing the Cre gene and confirmed that EGFP mRNA was transcribed in the virus-injected larvae.     

Alpha beta T cell receptor and CD3 transduce different signals in immature T cells. Implications for selection and tolerance

Ligand binding to alpha beta TCR has different consequences in thymocytes at different developmental stages, causing alternatively positive selection, clonal deletion, or activation. These various functional consequences may be due to changes in the signaling properties of the receptor complex during development. In this report we show that alpha beta TCR engagement on immature thymocytes has different effects on intracellular free calcium concentrations than alpha beta TCR engagement on mature T cells. In contrast, CD3 engagement on immature thymocytes and mature T cells has the same effect on intracellular free calcium, suggesting that altered signal transduction in immature thymocytes may be due to inefficient alpha beta TCR-CD3 coupling. These studies also suggest that in certain T cell populations, activation events resulting from ligation of CD3 may not accurately reflect the activation events resulting from ligation of the physiologic receptor, alpha beta TCR.     

Thy-1-positive cells in the subodontoblastic layer possess high potential to differentiate into hard tissue-forming cells

The cells of the subodontoblastic cell-rich layer in dental pulp are speculated to contain odontoblast progenitor cells because of their positional relationship with odontoblasts as well as their high alkaline phosphatase (ALP) activity. However, it has yet to be determined whether these cells have the ability to differentiate into odontoblastic cells. In the present study, we firstly found that the majority of cells in the subodontoblastic layer expressed Thy-1, a cell-surface marker of stem and progenitor cells. Then, we evaluated the capacity of Thy-1 high- and low-expressing (Thy-1(high) and Thy-1(low)) cells separated from rat dental pulp cells by use of a fluorescence-activated cell sorter to differentiate into hard tissue-forming cells in vitro and in vivo. Following stimulation with bone morphogenetic protein-2, Thy-1(high) cells in vitro showed accelerated induction of ALP activity and formation of alizarin red-positive mineralized matrix compared with Thy-1(low) cells. Furthermore, subcutaneous implantation of Thy-1(high) cells efficiently induced the formation of bone-like matrix. These results collectively suggest that Thy-1-positive dental pulp cells localized in the subodontoblastic layer had the ability to differentiate into hard tissue-forming cells, and thus these cells may serve as a source of odontoblastic cells.     

Activity dynamics and propagation of synchronous spiking in locally connected random networks

Random network models have been a popular tool for investigating cortical network dynamics. On the scale of roughly a cubic millimeter of cortex, containing about 100,000 neurons, cortical anatomy suggests a more realistic architecture. In this locally connected random network, the connection probability decreases in a Gaussian fashion with the distance between neurons. Here we present three main results from a simulation study of the activity dynamics in such networks. First, for a broad range of parameters these dynamics exhibit a stationary state of asynchronous network activity with irregular single-neuron spiking. This state can be used as a realistic model of ongoing network activity. Parametric dependence of this state and the nature of the network dynamics in other regimes are described. Second, a synchronous excitatory stimulus to a fraction of the neurons results in a strong activity response that easily dominates the network dynamics. And third, due to that activity response an embedding of a divergent-convergent feed-forward subnetwork (as in synfire chains) does not naturally lead to a stable propagation of synchronous activity in the subnetwork; this is in contrast to our earlier findings in isolated subnetworks of that type. Possible mechanisms for stabilizing the interplay of volleys of synchronous spikes and network dynamics by specific learning rules or generalizations of the subnetworks are discussed.     

Efficient Protein Expression in <Emphasis Type="Italic">Bombyx mori</Emphasis> Larvae of the Strain d17 Highly Sensitive to <Emphasis Type="Italic">B. mori</Emphasis> Nucleopolyhedrovirus

The recombinant protein expression by Bombyx mori nucleopolyhedrovirus (BmNPV) infecting silkworm larvae or pupae may endow us with a potent system for the production of large eukaryotic proteins. However, the screening of silkworm strains ideally suited to this method has scarcely been conducted. In the present study, we injected recombinant BmNPV containing a reporter gene, luciferase or DsRed, into hemocoel of fifth instar larvae of selected 12 silkworm strains. Among them, the strain d17 is found to be the highest in reporter expression from the intrinsic polyhedrin promoter of Autographa californica NPV or the silkworm actin A3 promoter. These results suggest that the d17 strain is highly permissive for BmNPV replication and is the most likely candidate of a “factory” for large-scale expression using the BmNPV bacmid system.     

Site-directed mutagenesis by biolistic transformation efficiently generates inheritable mutations in a targeted locus in soybean somatic embryos and transgene-free descendants in the T1 generation

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is being rapidly developed for mutagenesis in higher plants. Ideally, foreign DNA introduced by this system is removed in the breeding of edible crops and vegetables. Here, we report an efficient generation of Cas9-free mutants lacking an allergenic gene, Gly m Bd 30K, using biolistic transformation and the CRISPR/Cas9 system. Five transgenic embryo lines were selected on the basis of hygromycin resistance. Cleaved amplified polymorphic sequence analysis detected only two different mutations in e all of the lines. These results indicate that mutations were induced in the target gene immediately after the delivery of the exogenous gene into the embryo cells. Soybean plantlets (T₀ plants) were regenerated from two of the transgenic embryo lines. The segregation pattern of the Cas9 gene in the T₁ generation, which included Cas9-free plants, revealed that a single copy number of transgene was integrated in both lines. Immunoblot analysis demonstrated that no Gly m Bd 30K protein accumulated in the Cas9-free plants. Gene expression analysis indicated that nonsense mRNA decay might have occurred in mature mutant seeds. Due to the efficient induction of inheritable mutations and the low integrated transgene copy number in the T₀ plants, we could remove foreign DNA easily by genetic segregation in the T₁ generation. Our results demonstrate that biolistic transformation of soybean embryos is useful for CRISPR/Cas9-mediated site-directed mutagenesis of soybean for human consumption.