Direct and indirect effects of recombinant human granulocyte-colony stimulating factor on in vitro colony formation of human bladder cancer cells

Although the present experimental use of recombinant human granulocyte-colony-stimulating factor (rG-CSF) has been proven to alleviate the myelosuppression induced by antitumor chemotherapy, it is also believed to stimulate growth of some nonhematopoietic tumor cells. We investigated both the direct and indirect effects of rG-CSF on in vitro colony formation of human bladder cancer cell lines using a modified human tumor clonogenic assay. Peripheral blood mononuclear cells (PBMC) were used as feeder cells (a mixture of 5×10⁴ monocytes/dish and 5×10⁵ lymphocytes/dish obtained from healthy donors). Human bladder cancer cell lines KK-47, TCCSUP and T24, all derived from human transitional-cell carcinomas, were incubated continuously with various concentrations of rG-CSF ranging from 0.01 ng/ml to 10 ng/ml both with and without PBMC for 7–21 days. The concentrations of rG-CSF used were chosen as being in the range of achievable serum concentrations in patients treated with rG-CSF. At the end of incubation, colonies were counted under an inverted phase-contrast microscope, and an increase in the number of colonies in comparison with the control was used to evaluate the effects of rG-CSF. Results were expressed as a percentage of controls. rG-CSF in the upper layer at concentrations ranging from 0.1 ng/ml to 10 ng/ml stimulated the colony formation of all the cancer cell lines tested in the absence of PBMC in the feeder layer, whereas cells with PBMC in the feeder layer were significantly stimulated more than those without PBMC in the feeder layer (P<0.05) up to a certain concentration, which varied from cell line to cell line. At higher concentrations of rG-CSF, no further stimulation but, on the contrary, a decrease in colony formation was observed in cells with PBMC in the feeder layer in all the cell lines tested. Colony formation in KK-47 and T24 cell lines was significantly inhibited at 5 ng/ml and/or 10 ng/ml rG-CSF compared with cells without PBMC in the feeder layer. Our results suggest that rG-CSF may have both direct and indirect stimulatory effects on the growth of human bladder cancer cell lines in vitro. The results obtained also raise the possibility of adverse effects of rG-CSF in bladder cancer patients whose malignant cells may be directly and indirectly stimulated by this factor while it is being used clinically to alleviate the myelosuppression induced by antitumor chemotherapy.     

Reversible susceptibility to natural cell-mediated cytotoxicity observed in altered BHK cells resistant to HVJ (Sendai virus) infection

Altered baby hamster kidney (BHK-R) cells which were subcultured in the continuous presence of HVJ (hemagglutinating virus of Japan--the Sendai strain of parainfluenza 1 virus) showed a high susceptibility to natural cell-mediated cytotoxicity, although BHK-R cells are not transiently or persistently infected with HVJ but contain the restricted amount of sialic acid. By repeated subcultivation of BHK-R cells in growth medium free of HVJ, the sensitivity to natural killer cytotoxicity decreased to the level of normal BHK cells with a counter increase of cellular sialic acid, and the subsequent treatment of the cells with neuraminidase caused a loss of proper sialic acid residues, once again resulting in a significant enhancement of lysis by natural killer cells. In the BHK-R cell system which exhibits a reversible resistance to the interferon action, the enhancing effect induced by interferon on target cell susceptibility to natural killer activity became more pronounced in accord with the recovery of sensitivity to the antiviral action of interferon.     

Monitoring oxygen consumption of single mouse embryos using an integrated electrochemical microdevice

Oxygen consumption (respiration activity) has been found to be the most remarkable criterion for determining the viability of an embryo produced in vitro. In this study, we propose an accurate, simple, and user-friendly device for measurement of the oxygen consumption of single mammalian embryos. An integrated electrode array was fabricated to determine the oxygen consumption of a single embryo, including the blastocyst stage, which has an inhomogeneous oxygen consumption rate, using a single measurement procedure. A single mouse embryo was positioned in a microwell at the center of an integrated electrode array, using a mouthpiece pipette, and immobilized by a cylindrical micropit with good reproducibility. The oxygen consumption of two-cell, morula, and blastocyst stages was measured amperometrically using the device. The recorded current profile was corrected to take into consideration transient background current during the measurement. A calculation method for oxygen consumption based on spherical diffusion centered on the defined point of the device was developed. This procedure is quite simple because it is not necessary to estimate the radius of the embryo being measured. The calculated values of oxygen consumption for two-cell, morula, and blastocyst stages were 1.36 ± 0.33 × 10⁻¹⁵ mol s⁻¹, 1.38 ± 0.58 × 10⁻¹⁵ mol s⁻¹, and 3.44 ± 2.07 × 10⁻¹⁵ mol s⁻¹, respectively. The increasing pattern of oxygen consumption from morula to blastocyst agreed well with measurements obtained using conventional scanning electrochemical microscopy (SECM).     

Proposal of leukotoxin, 9,10-epoxy-12-octadecenoate, as a burn toxin

It is postulated that toxic substances (burn toxin) synthesized in burned skin are transferred into general circulation and cause multiple organ failure. We found a highly cytotoxic substance, leukotoxin, a linoleate epoxide, exists in burned skin. Leukotoxin, as the name indicates, was synthesized by leukocytes from linoleate as a substrate. The aim of this study is to evaluate the possibility of leukotoxin as a burn toxin. We studied plasma leukotoxin level of four patients with extensive burns (over 50% of body surface area) and examined coagulation studies in these patients. We detected considerable amounts of leukotoxin (11.4 nmol/ml-37.0 nmol/ml) in all patients. Leukotoxin was not detected in the control subjects. Pulmonary edema, cardiac failure, and coagulation abnormalities were found in these patients. Exogeneously administered leukotoxin induced similar pathological conditions in experimental animals to those observed in patients with extensive burns. Hence, it is concluded that leukotoxin is a responsible substance as a burn toxin.     

Trail traffic flow prediction by contact frequency among individual ants

Ants build a trail that leads to a new location when they move their colony. The trail’s traffic flows smoothly, regardless of the density on the trail. To the best of our knowledge, such a phenomenon has been reported only for ant species. The trail’s capacity is known as trail traffic flow. In this paper, we propose a probabilistic model of trail traffic flow, which overcomes some inadequacies of the kinetic model previously proposed in the literature. Our model answers a question unsolved by the previous model, namely, how many worker ants form such a density-independent trail. We focus on ants’ responses to mutual contacts that involve individuals in trail formation. We propose a model in which contact frequency predicts the number of worker ants that form a trail. We verify that our model’s estimates match the empirical data that ant experts reported in the literature. In modeling and evaluation, we discuss an intelligent ant species, the house-hunting ant Temnothorax albipennis, which is popular among the ant experts.     

Optimization of 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidines to generate a highly selective PI3Kδ inhibitor

Chemical optimization of the 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine (THPP) scaffold was conducted with a focus on cellular potency while maintaining high selectivity against PI3K isoforms. Compound 11f was identified as a potent, highly selective and orally available PI3Kδ inhibitor. In addition, 11f exhibited efficacy in an in vivo antibody production model. The desirable drug-like properties and in vivo efficacy of 11f suggest its potential as a drug candidate for the treatment of autoimmune diseases and leukocyte malignancies.     

Long-term changes in temperate marine fish assemblages are driven by a small subset of species

The species composition of plant and animal assemblages across the globe has changed substantially over the past century. How do the dynamics of individual species cause this change? We classified species into seven unique categories of temporal dynamics based on the ordered sequence of presences and absences that each species contributes to an assemblage time series. We applied this framework to 14,434 species trajectories comprising 280 assemblages of temperate marine fishes surveyed annually for 20 or more years. Although 90% of the assemblages diverged in species composition from the baseline year, this compositional change was largely driven by only 8% of the species' trajectories. Quantifying the reorganization of assemblages based on species shared temporal dynamics should facilitate the task of monitoring and restoring biodiversity. We suggest ways in which our framework could provide informative measures of compositional change, as well as leverage future research on pattern and process in ecological systems.     

Cloning and Sequencing of the Spore Germination Gene of Bacillus megaterium ATCC 12872: Similarities to the NaH-Antiporter Gene of Enterococcus hirae

The germination mutant TM-31 of Bacillus megaterium ATCC 12872, was isolated by transposon Tn917 insertional mutagenesis. Glucose, L -proline, L -leucine and KNO₃ germinated TM-31 poorly. The DNA in the region of the Tn917 insertion was cloned, and its nucleotide sequence determined. One major open reading frame was present on the cloned DNA. The hydrophobic protein encoded is presumably membrane-associated. A homology search revealed that the gene encoded in the region of the Tn917 insertion is homologous to napA of Enterococcus hirae. napA codes for the NaH-antiporter. It is hypothesized that transport of cations must play an important role in spore germination in B. megaterium ATCC 12872.     

Integrated Proteomics Identified Up-Regulated Focal Adhesion-Mediated Proteins in Human Squamous Cell Carcinoma in an Orthotopic Murine Model

Understanding the molecular mechanisms of oral carcinogenesis will yield important advances in diagnostics, prognostics, effective treatment, and outcome of oral cancer. Hence, in this study we have investigated the proteomic and peptidomic profiles by combining an orthotopic murine model of oral squamous cell carcinoma (OSCC), mass spectrometry-based proteomics and biological network analysis. Our results indicated the up-regulation of proteins involved in actin cytoskeleton organization and cell-cell junction assembly events and their expression was validated in human OSCC tissues. In addition, the functional relevance of talin-1 in OSCC adhesion, migration and invasion was demonstrated. Taken together, this study identified specific processes deregulated in oral cancer and provided novel refined OSCC-targeting molecules.     

Mammalian CNTD1 is critical for meiotic crossover maturation and deselection of excess precrossover sites

Meiotic crossovers (COs) are crucial for ensuring accurate homologous chromosome segregation during meiosis I. Because the double-strand breaks (DSBs) that initiate meiotic recombination greatly outnumber eventual COs, this process requires exquisite regulation to narrow down the pool of DSB intermediates that may form COs. In this paper, we identify a cyclin-related protein, CNTD1, as a critical mediator of this process. Disruption of Cntd1 results in failure to localize CO-specific factors MutLγ and HEI10 at designated CO sites and also leads to prolonged high levels of pre-CO intermediates marked by MutSγ and RNF212. These data show that maturation of COs is intimately coupled to deselection of excess pre-CO sites to yield a limited number of COs and that CNTD1 coordinates these processes by regulating the association between the RING finger proteins HEI10 and RNF212 and components of the CO machinery.