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).     

Mechanisms for the spontaneous formation of covalently linked polymers of the terminal membranolytic complement protein (C9)

Purified human C9 spontaneously polymerizes upon prolonged incubation at 37 degrees C, and a fraction of these C9 polymers becomes resistant to dissociation by sodium dodecyl sulfate (SDS) and reducing agents. We examined possible mechanisms for this spontaneous covalent linking of C9. The following results are consistent with the conclusion that the formation of the covalently linked C9 polymer involves disulfide linking. 1) In addition to the SDS/dithiothreitol (DTT)-resistant C9 polymer (Mr = 950,000), disulfide-linked C9 dimers and trimers were formed upon incubation of C9 at 37 degrees C for 64 h. 2) The C9 polymer formed upon incubation at 37 degrees C for 64 h was resistant to dissociation by 6 M guanidine hydrochloride, 20 mM DTT but was dissociated by 6 M guanidine thiocyanate alone, yielding disulfide-linked C9 oligomers. 3) The formation of the SDS/DTT-resistant C9 polymer was completely inhibited by 1 mM iodoacetamide and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), while DTNB enhanced the formation of disulfide-linked C9 oligomers. 4) A significant amount of free sulfhydryl group was detected in the polymerized C9 samples with various SH-specific reagents, though native C9 reacted with none of these reagents. In addition, inhibition by 1 mM iodoacetamide of C9 disulfide linking inhibited the self-association of C9 as analyzed by gel filtration on TSK-G4000 SW, whereas enhancement by 1mM DTNB of C9 disulfide linking enhanced C9 self-association. Thus, these results indicate that C9 disulfide linking that occurs upon C9 polymerization is an intrinsic property of C9 which is of importance in the formation of the stable C9 polymer structure.     

Some KpnI family members are associated with the Alu family in the human genome

The structures of the termini and their flanking regions of two human KpnI family members were investigated. The two differed in length, but the starting sequence at one terminal (defined as the 5' terminal) was found to be common to both members. The Alu family sequence was found in the 5' flanking regions. The KpnI family sequence started several base-pairs downstream from the 3' end of the Alu family sequence. In both cases, the Alu family sequence was not flanked by the direct repeat sequence common to the Alu family. These two members showed no sequence homology in 3' terminal regions. Interestingly, the Alu family plus the KpnI family unit was found to be flanked by a direct repeat sequence of several base-pair length. Based on these findings, relationship between the Alu family and KpnI family is discussed.     

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.     

Origins of laboratory mice deduced from restriction patterns of mitochondrial DNA

To determine the origins of laboratory mice, the restriction patterns of mitochondrial DNAs (mtDNAs) from various strains were compared with those of relevant subspecies and/or races of Mus musculus. In most strains and substrains of laboratory mice examined (50/55), the cleavage patterns were identical to those of the European subspecies M. m. domesticus. Those that varied include two sublines of NZB, the strain NZC, and the Japanese strain RR. The NZB and NZC patterns were identical to that of the European subspecies M. m. brevirostris, which itself has restriction patterns similar to M. m. domesticus. On the other hand, the RR pattern was identical to M. m. molossinus-like mice trapped in Western China and slightly different from Japanese M. m. molossinus. These findings suggest that the strains NZB and NZC stemmed from a European founder stock which differed from the ancestral stocks of other laboratory strains and that the ancestral mice of the RR strain had been transported from China to Japan. Therefore, most laboratory strains of mice are derived from the European subspecies M. m. domesticus while M. m. brevirostris and M. m. molossinus have made minor contributions. M. m. musculus does not appear to have made any contribution.