Isolation and Sequencing of a Genomic Clone for Mouse Brain Specific Small RNA

We isolated a mouse genomic clone that hybridized with small RNA present in the cytoplasm of the brain. The RNA was about 150 nucleotides long. This RNA seemed to be specific to the brain, since it was not found in the liver or kidney. The clone DNA contained a sequence homologous to 82-nucleotide "identifier" core sequence of cDNA clones of rat. The sequence contained a split promoter for RNA polymerase III and was flanked by a 12-nucleotide direct repeat (ATAAATAATTTA).     

A radioimmunoassay for'plasma progesterone

A radioimmunoassay for progesterone was developed which uses one micro-column chromatography for purification of hexane extracts of plasma.     

Global bifurcation structure of a Bonhoeffer-van der Pol oscillator driven by periodic pulse trains

The Bonhoeffer-van der Pol (BVP) oscillator is a valuable dynamical system model of pacemaker neurons. Isochrons, phase transition curves (PTC), and two dimensional bifurcation diagrams served to analyze the neuron's response to periodic pulse stimuli. Responses are described and explained in terms of the nonlinear dynamical system theory. An important issue in the generation of spikes by pacemaker neurons is the existence of both slow and fast dynamics in the state point's trajectory in the phase plane. It is this feature in particular that makes the BVP oscillator a faithful model of living pacemaker neurons. Comparison of the model's responses with those of a living pacemaker was based also on return maps of interspike intervals. Analyzed in detail were the complex discharges called stammering which involve interspike intervals that arise unpredictably and exhibit histograms with several modes separated by the equal intervals.Supported by Trent H. Wells Jr. Inc.     

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     

Determination of the NMR solution structure of a specific DNA complex of the Myb DNA-binding domain

Summary The solution structure of a specific DNA complex of the minimum DNA-binding domain of the mouse c-Myb protein was determined by distance geometry calculations using a set of 1732 nuclear Overhauser enhancement (NOE) distance restraints. In order to determine the complex structure independent of the initial guess, we have developed two different procedures for the docking calculation using simulated annealing in four-dimensional space (4D-SA). One is a multiple-step procedure, where the protein and the DNA were first constructed independently by 4D-SA using only the individual intramolecular NOE distance restraints. Here, the initial structure of the protein was a random coil and that of the DNA was a typical B-form duplex. Then, as the starting structure for the next docking procedure, the converged protein and DNA structures were placed in random molecular orientations, separated by 50 Å. The two molecules were docked by 4D-SA utilizing all the restraints, including the additional 66 intermolecular distance restraints. The second procedure comprised a single step, in which a random-coil protein and a typical B-form DNA duplex were first placed 70 Å from each other. Then, using all the intramolecular and intermolecular NOE distance restraints, the complex structure was constructed by 4D-SA. Both procedures yielded the converged complex structures with similar quality and structural divergence, but the multiple-step procedure has much better convergence power than the single-step procedure. A model study of the two procedures was performed to confirm the structural quality, depending upon the number of intermolecular distance restraints, using the X-ray structure of the engrailed homeodomain-DNA complex.Abbreviations rmsd root-mean-square deviation - NOE nuclear Overhauser enhancement - 4D-SA simulated annealing in four-dimensional space - Myb-R2R3 repeats 2 and 3 of the DNA-binding domain of the c-Myb protein - DNA 16 Myb-specific binding DNA duplex with 16 base pairs - IHDD-C residues 3 to 59 of the C-chain of the engrailed homeodomain-DNA complex - DNA11 DNA duplex with base pairs 9 to 19 of the engrailed homeodomain-DNA complex     

Calcium dependent action potentials in skeletal muscle fibres of a beetle larva, Xylotrupes dichotomus

The ionic requirement for generating action potentials in ventral longitudinal muscle fibers dissected from beetle larvae was examined by conventional electrophysiological techniques. Muscle fibers that generated only graded responses in physiological saline were able to generate an all-or-none action potential when the potassium permeability of the membrane was inhibited by tetraethylammonium⁺ added to the saline. The peak of the action potential thus elicited was intimately related to the external Ca⁺⁺ concentration. The action potential was blocked by Co⁺⁺ which is known as a competitive inhibitor of Ca-spikes. Neither tetrodotoxin (3 μM) nor a Na-free condition effectively blocked the generation of the action potential. Mg⁺⁺ induced a shift in the peak of the action potential; this was, however, due to the stabilizing action of Mg⁺⁺ but not due to the penetration of Mg⁺⁺ through the muscle membrane. No action potential was elicited in the muscle fiber when immersed in a Ca-free, EGTA saline even when a high concentration of either Mg⁺⁺, Na⁺, or tetraethylammonium⁺ was present. The action potential of the larval muscle fiber was thus concluded to be a Ca-spike, through the channel of which Na⁺ or Mg⁺⁺ did not penetrate.     

Identification of some abnormal metabolites in psoriatic nail using gas chromatography—mass spectrometry

A gas chromatographic—mass spectrometric analysis was used to separate and identify abnormal compounds in the nail of psoriatic patients. The nail was extracted with heated ethanol, and the extract was analyzed with and without trimethylsilylation. Tetradecanoic acid octadecyl ester, hexadecanoic acid octadecyl ester and octadecanoic acid octadecyl ester were first identified in the psoriatic nail, but were not detected in normal nail.     

Delineation of the mechanism of inhibition of human T cell activation by PGE2

The capacity of PGE2 to inhibit human T cell responses was examined by investigating its effect on mitogen-induced IL-2 production and proliferation of highly purified CD4+ T cells. PGE2 inhibited both PHA and anti-CD3 induced proliferation and IL-2 production by an action directly on the responding T cell. Inhibition of IL-2 production reflected decreased accumulation of mRNA for IL-2. A variety of other cAMP elevating agents exerted similar inhibitory effects. Inhibition of proliferation could be overcome by supplemental IL-2, PMA, or the anti-CD28 mAb 9.3. Although PMA and 9.3 markedly increased the amount of IL-2 produced by mitogen-stimulated T cells, the percentage inhibition of IL-2 secretion caused by PGE2 and other cAMP elevating agents remained comparable in these costimulated cultures. Rescue of T cell DNA synthesis by these agents appeared to reflect the finding that, although PGE2 markedly inhibited IL-2 production, the absolute amount of IL-2 produced was increased sufficiently to sustain mitogen-induced proliferation. As anticipated, PGE2, forskolin, and cholera toxin increased T cell cAMP levels. The quantity of cellular cAMP generated in response to PGE2, cholera toxin, and forskolin could be inhibited by PMA or 2',5'-dideoxyadenosine. Using these reagents, the inhibitory effects of PGE2 were found to reflect intracellular cAMP levels, but only within a very narrow range. The results indicate that by elevating cAMP levels, PGE2 inhibits human T cell IL-2 production at a point that is common to both the CD3 and CD28 signaling pathways.