Enhancement of cell growth by intracellularly expressed herpes simplex virus thymidine kinase

A recombinant cell line (NIH3T3:pLtkSN) was made by infecting parental cells (NIH3T3) with a recombinant retrovirus (pLtkSN) encoding herpes simplex virus thymidine kinase (HSVtk) gene. The cells expressing HSVtk (NIH3T3:pLtkSN) grew 2.3 times more than the parental cells (NIH3T3) in Dulbecco's Modified Eagles Media containing 10% (v/v) horse serum. The NIH3T3:pLtkSN cells also showed a significant enhancement in the maximal cell concentration and the specific growth rate even at 2.5% serum concentration. The specific O2 uptake rate of NIH3T3 was 2.1 times greater than that of NIH3T3:pLtkSN. Under both O2-limited and O2-unlimited conditions, it appears that HSVtk plays an important role in enhancing the growth characteristics of animal cells.     

Cultivation of mammalian cells as aggregates in bioreactors: effect of calcium concentration of spatial distribution of viability

Recombinant human kidney epithelial 293 cells were cultivated as aggregates in suspension. The concentration calcium ion, in the range of 100 muM to 1mM, affected the rate of aggregate formation. During the course of cultivation the size distribution of aggregates shifted and the fraction of larger aggregates increased. This effect was more profound in cultures with a high calcium concentration. Scanning and transmission microscopic examination of the aggregates revealed that cell packing was greater in the high calcium cultures and that ultrastructural integrity was retained in aggregates from both low and high calcium cultures. Confocal microscopy was applied to examine the viability of cells in the interior of the aggregates. High viability was observed in the aggregates obtained from exponentially growing cultures. Aggregates from the high calcium culture in the stationary phase exhibited lower viability in the interior. With its ease of retention in a perfusion bioreactor, aggregate cultures offer an alternative choice for large-scale operation. (c) 1993 John Wiley & Sons, Inc.     

Solid cultures of thrips-pathogenic fungi Isaria javanica strains for enhanced conidial productivity and thermotolerance

Entomopathogenic fungi have great potential to control agricultural and horticultural insect pests, however optimizing conidial production systems to demonstrate high productivity and stability still needs additional efforts for successful field application and industrialization. Although many virulent entomopathogenic fungal isolates have been viewed as potential candidates in a laboratory environment, very few of the isolates are being used in practice for application in agricultural fields as commercial products. I. javanicus is an entomopathogenic fungus that is parasitic to various diverse coleopteran and lepidopteran insects and thought good candidate as biopesticdes. In this work, the basic characteristics of two entomopathogenic fungi, I. javanica FG340 and Pf04, were investigated in morphological examinations, genetic identification, and virulence against Thrips palmi, and then the feasibility of various grains substrates for conidial production was assessed, particularly focusing on conidial productivity and thermotolerance. Isaria javanica FG340 and Pf04 conidia were solid-cultured on 12 grains for 14?days in a Petri dish. Of the tested Italian millet, perilla seed, millet and barley-based cultures showed high conidial production. The four-grain media yielded >1?×?10⁹ conidia/g of I. javanica FG340 and Pf04. Pf04 strain had enhanced thermotolerance up to 45?°C when cultured on Italian millet. In application, it was easy to make a conidial suspension using the cultured grains, and several surfactants were tested to release the conidia. This work suggests several possible inexpensive grain substrates by which to promote conidial production combined with enhanced stability against exposure to high temperature.     

The yeast Tor signaling pathway is involved in G2/M transition via polo-kinase

The target of rapamycin (Tor) protein plays central roles in cell growth. Rapamycin inhibits cell growth and promotes cell cycle arrest at G1 (G0). However, little is known about whether Tor is involved in other stages of the cell division cycle. Here we report that the rapamycin-sensitive Tor complex 1 (TORC1) is involved in G2/M transition in S. cerevisiae. Strains carrying a temperature-sensitive allele of KOG1 (kog1-105) encoding an essential component of TORC1, as well as yeast cell treated with rapamycin show mitotic delay with prolonged G2. Overexpression of Cdc5, the yeast polo-like kinase, rescues the growth defect of kog1-105, and in turn, Cdc5 activity is attenuated in kog1-105 cells. The TORC1-Type2A phosphatase pathway mediates nucleocytoplasmic transport of Cdc5, which is prerequisite for its proper localization and function. The C-terminal polo-box domain of Cdc5 has an inhibitory role in nuclear translocation. Taken together, our results indicate a novel function of Tor in the regulation of cell cycle and proliferation.     

Discovery of 1-amino-4-phenylcyclohexane-1-carboxylic acid and its influence on agonist selectivity between human melanocortin-4 and -1 receptors in linear pentapeptides

Linear pentapeptides (Penta-cis-Apc-DPhe-Arg-Trp-Gly-NH2) containing 1-amino-4-phenylcyclohexane-1-carboxylic acid (cis-Apc) and substituted Apc are potent hMC4R agonists and they are inactive or weakly active in hMC1R, hMC3R, and hMC5R agonist assays. This study, together with our earlier report on 5-BrAtc, demonstrated the importance of replacing His6 with phenyl-containing rigid templates in achieving good hMC4R agonist potency and selectivity against hMC1R in linear pentapeptides.     

Vertical distribution of bacterial community is associated with the degree of soil organic matter decomposition in the active layer of moist acidic tundra

The increasing temperature in Arctic tundra deepens the active layer, which is the upper layer of permafrost soil that experiences repeated thawing and freezing. The increasing of soil temperature and the deepening of active layer seem to affect soil microbial communities. Therefore, information on soil microbial communities at various soil depths is essential to understand their potential responses to climate change in the active layer soil. We investigated the community structure of soil bacteria in the active layer from moist acidic tundra in Council, Alaska. We also interpreted their relationship with some relevant soil physicochemical characteristics along soil depth with a fine scale (5 cm depth interval). The bacterial community structure was found to change along soil depth. The relative abundances of Acidobacteria, Gammaproteobacteria, Planctomycetes, and candidate phylum WPS-2 rapidly decreased with soil depth, while those of Bacteroidetes, Chloroflexi, Gemmatimonadetes, and candidate AD3 rapidly increased. A structural shift was also found in the soil bacterial communities around 20 cm depth, where two organic (upper Oi and lower Oa) horizons are subdivided. The quality and the decomposition degree of organic matter might have influenced the bacterial community structure. Besides the organic matter quality, the vertical distribution of bacterial communities was also found to be related to soil pH and total phosphorus content. This study showed the vertical change of bacterial community in the active layer with a fine scale resolution and the possible influence of the quality of soil organic matter on shaping bacterial community structure.     

Use of moving aeration membrane bioreactor for the efficient production of tissue type plasminogen activator in serum free medium

A moving aeration-membrane (MAM) bioreactor was employed for the production of 2 μg/mL of tissue type Plasminogen Activator (tPA) in serum free medium from normal human fibroblast cells. This system could maintain high cell density for long periods of steady state conditions in perfusion cultivation. Under normal operating conditions, shear stress was as low as 0.65 dynes/cm² at the agitation speed of 80 rpm. Even though cell density gradually decreased with increasing agitation speed, tPA production increased linearly with increasing shear stress within a moderate range. This culture system allowed production of 2 μg tPA/mL while maintaining a high cell density of 1.0×10⁷ viable cells/mL.     

The protein-tyrosine kinase Syk interacts with the C-terminal region of tensin2

Syk is a 72-kDa protein-tyrosine kinase that regulates signaling through multiple cell surface receptors including those for antigens, immunoglobulins and proteins of the extracellular matrix. As part of its function, Syk binds a variety of downstream effectors through interactions that are often mediated by motifs that recognize phosphotyrosines. In a search for novel Syk-interacting proteins by yeast two-hybrid analysis, we identified tensin2 as a Syk-binding protein. Syk interacts with a fragment of tensin2 located near the C-terminus that contains SH2 and PTB domains. In epithelial cells, tensin2 localizes both to focal adhesions and to large cytoplasmic puncta. It is within these punctuate structures that Syk and tensin2 are co-localized. The clustering of Syk within these structures leads to its phosphorylation on tyrosine.     

Effect of Bacillus mesonae H20-5 Treatment on Rhizospheric Bacterial Community of Tomato Plants under Salinity Stress

Plant growth-promoting bacteria improve plant growth under abiotic stress conditions. However, their effects on microbial succession in the rhizosphere are poorly understood. In this study, the inoculants of Bacillus mesonae strain H20-5 were administered to tomato plants grown in soils with different salinity levels (EC of 2, 4, and 6 dS/m). The bacterial communities in the bulk and rhizosphere soils were examined 14 days after H20-5 treatment using Illumina MiSeq sequencing of the bacterial 16S rRNA gene. Although the abundance of H20-5 rapidly decreased in the bulk and rhizosphere soils, a shift in the bacterial community was observed following H20-5 treatment. The variation in bacterial communities due to H20-5 treatment was higher in the rhizosphere than in the bulk soils. Additionally, the bacterial species richness and diversity were greater in the H20-5 treated rhizosphere than in the control. The composition and structure of the bacterial communities varied with soil salinity levels, and those in the H20-5 treated rhizosphere soil were clustered. The members of Actinobacteria genera, including Kineosporia, Virgisporangium, Actinoplanes, Gaiella, Blastococcus, and Solirubrobacter, were enriched in the H20-5 treated rhizosphere soils. The microbial co-occurrence network of the bacterial community in the H20-5 treated rhizosphere soils had more modules and keystone taxa compared to the control. These findings revealed that the strain H20-5 induced systemic tolerance in tomato plants and influenced the diversity, composition, structure, and network of bacterial communities. The bacterial community in the H20-5 treated rhizosphere soils also appeared to be relatively stable to soil salinity changes.