Relationship between cell size and specific thrombopoietin productivity in chinese hamster ovary cells during dihydrofolate reductase-mediated gene amplification

When parental Chinese hamster ovary (CHO) cell clones that are capable of producing thrombopoietin (TPO) were subjected to high methotrexate (MTX) concentrations, clonal variations in cell growth were apparent. In the clones that had no significant enhancement in specific TPO productivity (q _Tpo) when a higher level of MTX was administered, their growth was not depressed significantly nor their cell size changed significantly. On the other hand, those clones that showed a significant enhancement inq _Tpo at higher a MTX dosage, cell growth was depressed initially but recovered during successive sub-cultures. Furthermore, their cell size increased, which suggested that changes in cell size may be indicative of an enhancedq _Tpo. When the enhancement of theq _Tpo of 9 clones after a high MTX dosage was plotted against the extent of the increase of their size, there was a linear correlation (r ²=0.80,P<0.001, ANOVA), which suggested that an enhancement ofq _Tpo after high MTX administration can be measured by the increase in their cell size. Taken together, our data demonstrate that the selection of amplified CHO cell clones with enhancedq _Tpo can be done based upon their increased size and growth pattern. This facilitates the development of highly productive recombinant CHO cell lines.     

Effects of polysaccharides from rhizomes of Curcuma zedoaria on macrophage functions.

The effects of Curcuma zedoaria, which is used as a condiment, in perfumery, and as a medicine, on immune response were investigated by measuring macrophage-stimulating activity in macrophages and RAW 264.7 cells. In this study, CZ-1 and CZ-1-III, the fractions partially purified from C. zedoaria, had a strong, dose-dependent lysosomal enzyme activity. It was suggested that active portions of CZ-1-III were polysaccharides rather than proteins. Phagocytic activity increased as a similar pattern in both the gram-negative and gram-positive bacteria, time-dependently. It was demonstrated that CZ-1-III can augment the oxygen burst response but had an even higher activity in vivo than in vitro. Also a significant increase of H2O2, NO, and TNF-alpha production was observed. However, the production of TNF-alpha at the concentration of 1,000 microg/ml decreased. These data suggested that C. zedoaria had macrophage-stimulating activity and the possibility of being used as a biological response modifier.     

Orexinergic Neurotransmission in Temperature Responses to Methamphetamine and Stress: Mathematical Modeling as a Data Assimilation Approach

Experimental Data

Orexinergic neurotransmission is involved in mediating temperature responses to methamphetamine (Meth). In experiments in rats, SB-334867 (SB), an antagonist of orexin receptors (OX1R), at a dose of 10 mg/kg decreases late temperature responses (t>60 min) to an intermediate dose of Meth (5 mg/kg). A higher dose of SB (30 mg/kg) attenuates temperature responses to low dose (1 mg/kg) of Meth and to stress. In contrast, it significantly exaggerates early responses (t<60 min) to intermediate and high doses (5 and 10 mg/kg) of Meth. As pretreatment with SB also inhibits temperature response to the stress of injection, traditional statistical analysis of temperature responses is difficult.

Mathematical Modeling

We have developed a mathematical model that explains the complexity of temperature responses to Meth as the interplay between excitatory and inhibitory nodes. We have extended the developed model to include the stress of manipulations and the effects of SB. Stress is synergistic with Meth on the action on excitatory node. Orexin receptors mediate an activation of on both excitatory and inhibitory nodes by low doses of Meth, but not on the node activated by high doses (HD). Exaggeration of early responses to high doses of Meth involves disinhibition: low dose of SB decreases tonic inhibition of HD and lowers the activation threshold, while the higher dose suppresses the inhibitory component. Using a modeling approach to data assimilation appears efficient in separating individual components of complex response with statistical analysis unachievable by traditional data processing methods.     

Paeonia lactiflora Enhances the Adhesion of Trophoblast to the Endometrium via Induction of Leukemia Inhibitory Factor Expression

In the present study, we investigated the role of Paeonia lactiflora Pall. extract on embryo implantation in vitro and in vivo. A polysaccharides depleted-water extract of P. lactiflora (PL-PP) increased LIF expression in human endometrial Ishikawa cells at non-cytotoxic doses. PL-PP significantly increased the adhesion of the human trophectoderm-derived JAr spheroids to endometrial Ishikawa cells. PL-PP-induced LIF expression was decreased in the presence of a p38 kinase inhibitor SB203580 and an MEK/ERK inhibitor U0126. Furthermore, endometrial LIF knockdown by shRNA reduced the expression of integrins β3 and β5 and adhesion of JAr spheroids to Ishikawa cells. In vivo administration of PL-PP restored the implantation of mouse blastocysts in a mifepristone-induced implantation failure mice model. Our results demonstrate that PL-PP increases LIF expression via the p38 and MEK/ERK pathways and favors trophoblast adhesion to endometrial cells.     

Simple atomistic modeling of dominant B m I n clusters in boron diffusion

In this paper, we present a simple atomistic model for describing the evolution of interstitial clusters during boron diffusion in kinetic Monte-Carlo (KMC) calculation. It has been known that clusters generated after ion implantation play a decisive role in the enhanced boron diffusion at the tail region while being immobile at the peak region. Our model, which is based on the simple continuum model, takes the intermediate clusters into account as well as dominant clusters for describing the evolutionary behavior of interstitial clusters during boron diffusion. We found that the intermediate clusters such as B₃I₃ and B₂I₃ play a significant role during the evolution of clusters despite the fact that the lifetimes of the corresponding intermediate clusters are relatively short due to low binding energies. Further, our investigation revealed that B₃I is the most dominant cluster after annealing. We applied our simple atomistic model to the study of boron retardation in arsenic pre-doped substrate. KMC simulation results were compared with experimental SIMS data, which supports our theoretical model.     

DNA Methyltransferase 1-associated Protein (DMAP1) Is a Co-repressor That Stimulates DNA Methylation Globally and Locally at Sites of Double Strand Break Repair

Correction of double strand DNA breaks proceeds in an error-free pathway of homologous recombination (HR), which can result in gene silencing of half of the DNA molecules caused by action by DNA methyltransferase 1 (DNMT1) (Cuozzo, C., Porcellini, A., Angrisano, T., Morano, A., Lee, B., Di Pardo, A., Messina, S., Iuliano, R., Fusco, A., Santillo, M. R., Muller, M. T., Chiariotti, L., Gottesman, M. E., and Avvedimento, E. V. (2007) PLoS Genet. 3, e110). To explore the mechanism that leads to HR-induced silencing, a genetic screen was carried out based on the silencing of a GFP reporter to identify potential partners. DMAP1, a DNMT1 interacting protein, was identified as a mediator of this process. DMAP1 is a potent activator of DNMT1 methylation in vitro, suggesting that DMAP1 is a co-repressor that supports the maintenance and de novo action of DNMT1. To examine critical roles for DMAP1 in vivo, lentiviral shRNA was used to conditionally reduce cellular DMAP1 levels. The shRNA transduced cells grew poorly and eventually ceased their growth. Analysis of the tumor suppressor gene p16 methylation status revealed a clear reduction in methylated CpGs in the shRNA cells, suggesting that reactivation of a tumor suppressor gene pathway caused the slow growth phenotype. Analysis of HR, using a fluorescence-based reporter, revealed that knocking down DMAP1 also caused hypomethylation of the DNA repair products following gene conversion. DMAP1 was selectively enriched in recombinant GFP chromatin based on chromatin immunoprecipitation analysis. The picture that emerges is that DMAP1 activates DNMT1 preferentially at sites of HR repair. Because DMAP1 depleted cells display enhanced HR, we conclude that it has additional roles in genomic stability.     

The Incidence of Non-tuberculous Mycobacterium Lung Disease in Patients with Suspected Pulmonary Tuberculosis

Non-tuberculous mycobacterium (NTM) lung disease is increasing in prevalence. We analyzed the frequency of NTM lung disease among patients who are suspected of tuberculosis. NTM was isolated from about one-fourth of the mycobacterium culture-positive patients and about half of these had NTM lung disease. Therefore, NTM isolates should be routinely identified at the species level for adequate treatment.     

Difference in microchip electrophoretic mobility between partially and fully PEGylated poly(amidoamine) dendrimers

The objective of this study was to investigate the difference in electrophoretic mobility between partially and fully poly(ethylene glycol)-conjugated poly(amidoamine) dendrimers (part-PEG-PAMAM and full-PEG-PAMAM, respectively) using a microchip capillary gel electrophoresis (MCGE). While MCGE allowed size-based separation of PEG-PAMAMs prepared with monomethoxy PEG-nitrophenyl carbonate, full-PEG-PAMAMs migrated slower than part-PEG-PAMAMs that were similar in size or larger. When the measured molecular weights obtained from MCGE analysis and the calculated molecular weights were plotted, each part-PEG-PAMAM and full-PEG-PAMAM showed correlation coefficients greater than 0.98. This study indicates that MCGE would be useful for characterizing PEG-PAMAMs with different PEGylation degrees.