Purification of rabbit tumor necrosis factor

Rabbit tumor necrosis factor (TNF) was purified and shown by SDS-PAGE to be a single protein of 18 kDa. TNF in 355 ml rabbit serum was precipitated with ammonium sulfate, and purified by repeated DEAE-Sephadex and Sephacryl S-200 chromatographies, and the final fractionation on Blue-Sepharose 6B. By this procedure its yield was 22% and its specific activity was 2.4 × 10⁷ U/mg protein. The sequence of the N-terminal 20 amino acids was determined.     

Krüppel-like is required for nonskeletogenic mesoderm specification in the sea urchin embryo

The canonical Wnt pathway plays a central role in specifying vegetal cell fate in sea urchin embryos. SpKrl has been cloned as a direct target of nuclear β-catenin. Using Hemicentrotus pulcherrimus embryos, here we show that HpKrl controls the specification of secondary mesenchyme cells (SMCs) through both cell-autonomous and non-autonomous means. Like SpKrl, HpKrl was activated in both micromere and macromere progenies. To examine the functions of HpKrl in each blastomere, we constructed chimeric embryos composed of blastomeres from control and morpholino-mediated HpKrl-knockdown embryos and analyzed the phenotypes of the chimeras. Micromere-swapping experiments showed that HpKrl is not involved in micromere specification, while micromere-deprivation assays indicated that macromeres require HpKrl for cell-autonomous specification. Transplantation of normal micromeres into a micromere-less host with morpholino revealed that macromeres are able to receive at least some micromere signals regardless of HpKrl function. From these observations, we propose that two distinct pathways of endomesoderm formation exist in macromeres, a Krl-dependent pathway and a Krl-independent pathway. The Krl-independent pathway may correspond to the Delta/Notch signaling pathway via GataE and Gcm. We suggest that Krl may be a downstream component of nuclear β-catenin required by macromeres for formation of more vegetal tissues, not as a member of the Delta/Notch pathway, but as a parallel effector of the signaling (Krl-dependent pathway).     

Damaged epithelia regenerated by bone marrow-derived cells in the human gastrointestinal tract

Studies have shown that bone marrow cells have the potential to differentiate into a variety of cell types. Here we show that bone marrow cells can repopulate the epithelia of the human gastrointestinal tract. Epithelial cells of male donor origin were distributed in every part of the gastrointestinal tract of female bone marrow transplant recipients. Donor-derived epithelial cells substantially repopulated the gastrointestinal tract during epithelial regeneration after graft-versus-host disease or ulcer formation. Regeneration of gastrointestinal epithelia with donor-derived cells in humans shows a potential clinical application of bone marrow-derived cells for repairing severely damaged epithelia, not only in the gastrointestinal tract but also in other tissues.     

Distance- and density-dependent seedling mortality caused by several diseases in eight tree species co-occurring in a temperate forest

To examine whether the Janzen–Connell mechanism applies to temperate forests, seedling survival and causes of mortality were investigated at two distances (beneath, far) from conspecific adults and at two densities (high, low) at each distance for seedlings (n = 7935) of eight tree species co-occurring in a hardwood forest. Six of the eight species showed distance- and/or density-dependent seedling mortality mainly caused by diseases and rodents. In four of the five species primarily killed by disease (i.e. damping-off, blight, rot, powdery mildew), the infectivity (probability of infection by the disease) and/or the virulence (proportion of seedlings killed to those infected by the disease) were higher beneath than far from conspecific adults. These findings suggest that host specificity and/or spatially heterogeneous activity of natural enemies play an important role in the reciprocal replacement of tree species, maintaining species diversity in temperate forests.     

Interaction between the Spatiotemporal Learning Rule (STLR) and Hebb type (HEBB) in single pyramidal cells in the hippocampal CA1 Area

The spatiotemporal learning rule (STLR), proposed as a non-Hebb type by Tsukada et al. (Neural Networks 9 (1996) 1357 and Tsukada and Pan (Biol. cyberm 92 (2005) 139), 2005), consists of two distinctive factors; “cooperative plasticity without a cell spike,” and “its temporal summation”. On the other hand, Hebb (The organization of behavior. John Wiley, New York, 1949) proposed the idea (HEBB) that synaptic modification is strengthened only if the pre- and post-cell are activated simultaneously. We have shown, experimentally, that both STLR and HEBB coexist in single pyramidal cells of the hippocampal CA1 area. The functional differences between STLR and HEBB in dendrite (local)-soma (global) interactions in single pyramidal cells of CA1 and the possibility of pattern separation, pattern completion and reinforcement learning were discussed.     

Amperometric dimethyl sulfoxide sensor using dimethyl sulfoxide reductase from Rhodobacter sphaeroides

An amperometric dimethyl sulfoxide (DMSO) sensor was constructed based on DMSO reductase (DMSO-R). DMSO-R from Rhodobacter sphaeroides f. sp. denitrificans was immobilized by BSA-glutaraldehyde cross-linking at the surface of a glassy carbon electrode. Mediators were added to the sample solution in a free form. Several mediators (methyl viologen (MV), benzyl viologen (BV), neutral red (NR), safranin T (ST), FMN, phenazine methosulfate (PMS)), which can donate electrons to DMSO-R, were examined with the DMSO-R immobilized electrode. Among them MV was selected as a model mediator because of its wide linear response range and fast response time. The response current was effected by the measurement temperature but hardly effected by the pH of the sample solution. The response current was increased with the measurement temperature up to 50 degrees C. A response current was observed at 1 microM DMSO and the response time was 20 s under the optimum conditions. The response was observed for approximately 2 weeks. By the reduction of Schiff base in the cross-linking layer the response range became narrower but most of the response current was retained at 300 microM of DMSO for more than 5 weeks.     

Purified Rabbit Interferon: Attempts to Demonstrate Interferon-Specific 3H-Protein

³H-protein present in highly purified preparations of rabbit interferon was not conclusively separated from noninterferon, cellular ¹⁴C-proteins by co-chromatography on carboxymethyl-Sephadex or by co-electrophoresis on acid or neutral polyacrylamide gels.     

Measurement of two- and three-bond 13C−1H J couplings to the Cδ carbons of leucine residues in staphylococcal nuclease

Summary A new ¹H-detected 3D NMR experiment is described that permits quantitative measurement of two- and three-bond ¹³C–¹H couplings in proteins with selectively ¹³C-enriched methyl sites. The method is demonstrated for staphylococcal nuclease selectively [5,5 ¹³C]-labeled in all 11 leucine positions and ligated with thymidine 3,5-biphosphate and Ca²⁺. Two- and three-bond ¹³C methyl-proton couplings are reported and, together with the measured three-bond J_CC in uniformly ¹³C-enriched staphylococcal nuclease, the ²- and the stereospecific assignments of the C methyl group with respect to the prochiral -protons were determined. The same residues that were previously found to have high degrees of internal mobility on the basis of ¹³C relaxation times have measured coupling constants that are indicative of motional averaging.     

Immobilization of Bacteria on to Polyester Cloth

Escherichia coli 3300 (constitutive for -galactosidase) had a greater adhesion (assayed by -galactosidase) to polyester cloth than to cotton cloth. Adhered bacteria developed immobilized cells on the cloth which can generate progeny efficiently. Similarly, four other Gram-negative bacteria and three Gram-positive bacteria had greater adhesion to polyester cloth than to cotton cloth. Since coating of polyester cloth with polyvinyl alcohol greatly decreased the bacterial adhesion, hydrophobic interaction is likely responsible for the bacterial adhesion to polyester cloth.