Pre-junctional inhibitory action of prostaglandin E2 on excitatory neuro-effector transmission in the human bronchus

The effects of prostaglandin E₂ (PGE₂) and indomethacin on excitatory neuro-effector transmission in the human bronchus were investigated by tension recording and microelectrode methods. PGE₂ (10⁻¹⁰–10⁻⁹M) suppressed the amplitude of twitch contractions and excitatory junction potentials (e.j.ps) evoked by field stimulation at a steady level of basal tension obtained by the combined application of indomethacin (10⁻⁵M) and FPL55712 (10⁻⁶M). In doses over 10⁻⁸M, PGE₂ reduced the muscle tone and dose-dependently suppressed the amplitude of twitch contractions. Indomethacin (10⁻⁵ or 5 × 10⁻⁵M) reduced the muscle tone and enhanced the amplitude of twitch contractions and e.j.ps evoked by field stimulation in the presence of FPL55712. PGE₂ (10⁻⁹M) had no effect on the post-junctional response of smooth muscle cells to exogenously applied acetylcholine (ACh) (4 × 10⁻⁷M). However, indomethacin (10⁻⁵M) significantly enhanced the ACh-induced contraction of the human bronchus. These results indicate that PGE₂ in low concentrations has a pre-junctional action to inhibit excitatory neuro-effector transmission in addition to a post-junctional action, presumably by suppressing transmitter release from the vagus nerve terminals in the human bronchial tissues.     

Identification of Mutations in the Pigment Cell-Specific Gene Located at the Brown Locus in Mouse

The pigment cell-specific gene, located at the brown (b)-locus in mouse, encodes the protein that determines the type of melanin synthesized. This protein is known as tyrosinase-related protein, but here we tentatively term it b-locus protein to avoid confusions with the related sequence cross-hybridizing to the tyrosinase gene. In order to identify the mutation at the b-locus, we have cloned and characterized the b-locus protein gene of BALB/c mouse (b/b, c/c). The gene is about 18 kb long and organized into 8 exons and 7 introns. Sequence analysis of the b-locus protein gene reveals four base changes within the protein-coding regions: two missense mutations and two silent mutations. Two missense mutations result in the Cys to Tyr substitution at position 86 (codon 110) and the Arg to His substitution at position 302 (codon 326) of a b-locus protein molecule. Using allele-specific amplification, we confirmed that these missense mutations are actually present in the genomic DNA of two b-mutant strains examined, BALB/c and DBA/2 (b/b, C/C) mice, suggesting that these mutations are specific for the mutant mice at the b-locus. Moreover, we are able to show that the b-locus protein containing Tyr 86 is not reactive with the anti-b-locus protein monoclonal antibody, TMH-1, in transient expression assays.     

Impairment of Mobility in Endodermal Cells by FAK Deficiency

Focal adhesion kinase (FAK) is a novel nonreceptor protein tyrosine kinase that localizes in focal adhesions. It is expressed in a variety of cell types, and we reported earlier that its deficiency causes a decrease of mobility in mesodermal cells with enhanced formation of focal adhesions. With embryoid bodies generated from embryonic stem cells, we also observed a decrease of mobility in FAK-deficient endodermal cells with enhanced focal adhesion formation.     

Effects of estradiol and testosterone on the synthesis,expression and degradation of androgen receptor in human uterine endometrial fibroblasts

The mechanism of known receptor-mediated androgen effects on the endometrial stroma was studied in endometrial fibroblasts derived from human uterus. 17-Estradiol (E) induced the expressions of androgen receptor (AR) mRNA, and predominantly increased the level of testosterone-binding sites (TBS) in uterine endometrial fibroblasts. The effect on the level of dihydrotestosterone-binding sites (DHTBS) was similar but smaller. This result suggests that the AR mRNA expressed might encode TBS, but probably not DHTBS. The TBS level increased by estrogen was down-regulated by testosterone (T) + E, but the AR mRNA expression increased by E was not down-regulated by E + T in the fibroblasts. Although the synthesis rate of AR was slightly increased (p<0.05) by E alone or E + T, the degradation rate of AR was significantly accelerated (p<0.05) by E + T in the fibroblasts. This result suggests that T might stimulate the metabolic rate of TBS, but does not inhibit the synthesis rate of AR mRNA to TBS in endometrial fibroblasts.     

A novel oncogene, ost, encodes a guanine nucleotide exchange factor that potentially links Rho and Rac signaling pathways.

Transfection of NIH3T3 cells with an osteosarcoma expression cDNA library led to the appearance of foci of morphologically transformed cells which were found to harbor a novel oncogene, ost. The ost product was activated by truncation of the N-terminal domain of the ost proto-oncogene and was highly tumorigenic in nude mouse assays. The proto-ost cDNA, isolated subsequently, encodes a predicted protein of 100 kDa containing DH (Db1 homology) and PH (pleckstrin homology) domains. Ost is mainly phosphorylated on serine and localized in the cytoplasm. Purified Ost protein catalyzed guanine nucleotide exchange on RhoA and Cdc42 among the Rho and Ras family members tested, indicating that Ost can activate these small GTP-binding proteins. Ost did not detectably associate with RhoA or Cdc42, but interacted specifically with the GTP-bound form of Rac1, suggesting that Ost can function as an effector of Rac1. These results suggest that Ost is a critical regulatory component which links pathways that signal through Rac1, RhoA and Cdc42. Of the tissues examined, expression of ost was the highest in brain and could be localized to neurons and alpha-tanycytes, suggesting that Ost may participate in axonal transport in these specialized cells.     

Activation of Xenopus Eggs by an Extract of Cynops Sperm

An extract obtained from Cynops sperm induced the activation of both Cynops and Xenopus eggs with accompanying changes in the potential of the egg membrane that were quite similar to those caused by the Cynops sperm. The activation-inducing properties of the extract were abolished by treatment with proteinase K or by heating (60°C, 15 min) and were associated with a protease activity against peptidyl Arg-MCA substrates. The activation of Xenopus eggs by the extract was inhibited by those substrates, or by protease inhibitors, aprotinin or leupeptin. The protease activity was localized in the acrosomal region of Cynops sperm. The activation of Xenopus eggs by the extract was prevented when the exterior concentration of Ca²⁺ions, [Ca²⁺]₀, was reduced to 1.5 μM, but it was enhanced when [Ca²⁺]₀ was increased to 340 μM. The activation of Xenopus eggs by the extract was not affected by positive clamping when [Ca²⁺]₀ was 340 μM. These results suggest that the sperm extract contains a protease that causes an increase in the influx of Ca²⁺ions that results in voltage-insensitive activation of the egg.     

Identification of Surface-Exposed Parts of Red-Light- and Far-Red-Light- Absorbing Forms of Native Pea Phytochrome by Limited Proteolysis

Peptide fragments were obtained by limited proteolysis withtrypsin and Staphylococcus aureus V 8 protease from either theP_R or the P_FR form of 121-kDa phytochrome purified from etiolatedpea (Pisum sativum L.) shoots. Patterns of bands after polyacrylamidegel electrophoresis in the presence of SDS of the digests weredifferent, with some bands appearing preferentially when thedigestions were carried out with the P_R or the P_FR form. Amino-terminalsequences of the fragments were analyzed to determine the exactlocations of the amino-termini of the fragments within the aminoacid sequence of the apoprotein of pea phytochrome. The aminoacid compositions of some of the sequenced fragments were determinedin order to confirm the carboxy-terminal amino acids. Threecleavage regions were identified as kinetically favored sitesof cleavage of P_FR (Arg-746 to Lys-752, around Glu-877 and aroundArg-1010), whereas only one was identified for P_R (Glu-38 toArg-62). Regions of Glu-255, Arg-383, Arg-583 to Glu-620 andLys-1093 to Glu-1115 were also identified as potential sitesof proteolytic cleavage in both forms of the phytochrome. Othercleavage sites, the specificities of which have not yet beendetermined, are Glu-404, Glu-695 and Lys-1045. Surface-exposed parts of phytochrome in the P_R and P_FR formsare discussed. (Received June 13, 1992; Accepted October 27, 1992)     

The synergistic effects of interleukin 2 and interleukin 7 on the proliferation and autologous tumor cell lysis of tumor-associated lymphocytes

Interleukin-7 (IL-7) has an ability to stimulate the proliferation of pre-B cells. It has been shown that IL-7 can also activate T lymphocytes. We here demonstrate that IL-7 in combination with interleukin-2 (IL-2) can drive cell proliferation and enhance the autologous tumor cell lysis by peripheral blood mononuclear cells (PBMC) and autologous mixed lymphocyte tumor cell culture (MLTC)-derived effector cells (MLTC cells). These synergistic effects of IL-2 and IL-7 on the proliferation and the augmentation of autologous tumor cell lysis were found for both effector cells. These effects were inhibited by neutralizing antibodies to IL-2 or IL-7, and by a combination of both antibodies, significantly. In terms of phenotypical expression, CD3 positive cells comprised the vast majority of MLTC cells after culture in medium containing IL-2 and IL-7 with an increase of IL-2 receptor positive cells.Abbreviations CD cluster differentiation - IFN interferon - IL interleukin - JRU Japanese Reference Unit - LAK lymphokine activated killer - mAb monoclonal antibody - MLTC mixed lymphocyte tumor cell culture - PBMC peripheral blood mononuclear cells - TILs tumor infiltrating lymphocytes     

The role of polypyrrole as charge transfer mediator and immobilization matrix for d-fructose dehydrogenase in a fructose sensor

A fructose dehydrogenase (FDH) modified electrode is produced by the electroadsorption of a layer of FDH on a platinum electrode followed by the electropolymerization of a polypyrrole (PPy) film around and over the enzyme. This immobilizes and stabilizes the enzyme as well as providing an electron transfer pathway to the electrode. The amperometric response to fructose and the enzymatic activity are measured as a function of PPy film thickness. The electrode is shown to have a maximum response at a PPy thickness of approximately the thickness of the enzyme layer. A measure of the electrode efficiency is also obtained, this is the amperometric response to fructose as a percentage of that expected on the basis of the enzyme activity. The functioning of the electrode is also dependent on the counter-ion used for PPy polymerization. This is shown to be mainly related to the nucleation and growth of the PPy film in the interfacial region.     

Transformation of Arabidopsis thaliana with the codA gene for choline oxidase; accumulation of glycinebetaine and enhanced tolerance to salt and cold stress

Glycinebetaine is one of the compatible solutes that accumulate in the chloroplasts of certain halotolerant plants when these plants are exposed to salt or cold stress. The codA gene for choline oxidase, the enzyme that converts choline into glycinebetaine, has previously been cloned from a soil bacterium, Arthrobacter globiformis. Transformation of Arabidopsis thaliana with the cloned codA gene under the control of the 35S promoter of cauliflower mosaic virus enabled the plant to accumulate glycinebetaine and enhanced its tolerance to salt and cold stress. At 300 mM NaCl, considerable proportions of seeds of transformed plants germinated well, whereas seeds of wild-type plants failed to germinate. At 100 mM NaCl, transformed plants grew well whereas wild-type plants did not do so. The transformed plants tolerated 200 mM NaCl, which was lethal to wild-type plants. After plants had been incubated with 400 mM NaCl for two days, the photosystem II activity of wild-type plants had almost completely disappeared, whereas that of transformed plants remained at more than 50% of the original level. When exposed to a low temperature in the light, leaves of wild-type plants exhibited symptoms of chlorosis, whereas those of transformed plants did not. These observations demonstrate that the genetic modification of Arabidopsis thaliana that allowed it to accumulate glycinebetaine enhanced its ability to tolerate salt and cold stress.