Developmental regulation of the alpha-mannosidase gene in Dictyostelium discoideum: control is at the level of transcription and is affected by cell density

1991 ◽  
Vol 11 (6) ◽  
pp. 3339-3347
Author(s):  
J Schatzle ◽  
A Rathi ◽  
M Clarke ◽  
J A Cardelli
Keyword(s):  
Cell Density ◽  
Dictyostelium Discoideum ◽  
High Cell Density ◽  
Specific Activity ◽  
Developmental Regulation ◽  
Cdna Probe ◽  
Mrna Levels ◽  
High Cell ◽  
Enzyme Specific Activity ◽  
Negative Mutant

In Dictyostelium discoideum, there is a group of genes that are expressed following starvation and when exponentially growing cells reach high densities. We have examined the expression of one of these genes, alpha-mannosidase. Using an alpha-mannosidase cDNA probe in Northern (RNA) blot analysis, we have shown that the previously observed increase in alpha-mannosidase enzyme-specific activity during development is due to an increase in the levels of alpha-mannosidase mRNA. mRNA levels reach a maximum by 8 h of development and then begin to decline by 14 to 22 h. Using nuclear run-on analysis, we have found that this gene is regulated at the level of transcription. We also examined the effects of cell-cell contacts, cyclic AMP levels, and protein synthesis on expression of this gene and found that they were not critical in regulating its expression. However, cell density did play a major role in the expression of alpha-mannosidase. High cell density or the presence of buffer conditioned by high-density cells was sufficient to induce expression of alpha-mannosidase, indicating that this is one of the prestarvation response genes. Finally, the alpha-mannosidase gene was not expressed in aggregation-negative mutant strain HMW 404.

scholarly journals Developmental regulation of the alpha-mannosidase gene in Dictyostelium discoideum: control is at the level of transcription and is affected by cell density.

1991 ◽  
Vol 11 (6) ◽  
pp. 3339-3347 ◽  
Author(s):  
J Schatzle ◽  
A Rathi ◽  
M Clarke ◽  
J A Cardelli
Keyword(s):  
Cell Density ◽  
Dictyostelium Discoideum ◽  
High Cell Density ◽  
Specific Activity ◽  
Developmental Regulation ◽  
Cdna Probe ◽  
Mrna Levels ◽  
High Cell ◽  
Enzyme Specific Activity ◽  
Negative Mutant

In Dictyostelium discoideum, there is a group of genes that are expressed following starvation and when exponentially growing cells reach high densities. We have examined the expression of one of these genes, alpha-mannosidase. Using an alpha-mannosidase cDNA probe in Northern (RNA) blot analysis, we have shown that the previously observed increase in alpha-mannosidase enzyme-specific activity during development is due to an increase in the levels of alpha-mannosidase mRNA. mRNA levels reach a maximum by 8 h of development and then begin to decline by 14 to 22 h. Using nuclear run-on analysis, we have found that this gene is regulated at the level of transcription. We also examined the effects of cell-cell contacts, cyclic AMP levels, and protein synthesis on expression of this gene and found that they were not critical in regulating its expression. However, cell density did play a major role in the expression of alpha-mannosidase. High cell density or the presence of buffer conditioned by high-density cells was sufficient to induce expression of alpha-mannosidase, indicating that this is one of the prestarvation response genes. Finally, the alpha-mannosidase gene was not expressed in aggregation-negative mutant strain HMW 404.

Role of procollagen mRNA levels in controlling the rate of procollagen synthesis

1983 ◽  
Vol 3 (2) ◽  
pp. 241-249
Author(s):  
L B Rowe ◽  
R I Schwarz
Keyword(s):  
Cell Density ◽  
High Cell Density ◽  
Control Point ◽  
Relative Increase ◽  
Limiting Factor ◽  
Mrna Levels ◽  
Primary Control ◽  
High Cell ◽  
Mrna Induction ◽  
Tendon Cells

Two factors must be present for primary avian tendon cells to commit 50% of their total protein production to procollagen: ascorbate and high cell density. Scorbutic primary avian tendon cells at high cell density (greater than 4 X 10(4) cells per cm2) responded to the addition of ascorbate by a sixfold increase in the rate of procollagen synthesis. The kinetics were biphasic, showing a slow increase during the first 12 h followed by a more rapid rise to a maximum after 36 to 48 h. In contrast, after ascorbate addition, the level of accumulated cytoplasmic procollagen mRNA (alpha 2) showed a 12-h lag followed by a slow linear increase requiring 60 to 72 h to reach full induction. At all stages of the induction process, the relative increase in the rate of procollagen synthesis over the uninduced state exceeded the relative increase in the accumulation of procollagen mRNA. A similar delay in mRNA induction was observed when the cells were grown in an ascorbate-containing medium but the cell density was allowed to increase. In all cases, the rate of procollagen synthesis peaked approximately 24 h before the maximum accumulation of procollagen mRNA. The kinetics for the increase in procollagen synthesis are not, therefore, in agreement with the simple model that mRNA levels are the rate-limiting factor in the collagen pathway. We propose that the primary control point is at a later step. Further support for this idea comes from inhibitor studies, using alpha, alpha'-dipyridyl to block ascorbate action. In the presence of 0.3 mM alpha, alpha'-dipyridyl there was a specific two- to threefold decrease in procollagen production after 4 h, but this was unaccompanied by a drop in procollagen mRNA levels. Therefore, inhibitor studies give further support to the idea that primary action of ascorbate is to release a post-translational block.

scholarly journals Reduced Levels of ATF-2 Predispose Mice to Mammary Tumors

2006 ◽  
Vol 27 (5) ◽  
pp. 1730-1744 ◽  
Author(s):  
Toshio Maekawa ◽  
Toshie Shinagawa ◽  
Yuji Sano ◽  
Takahiko Sakuma ◽  
Shintaro Nomura ◽  
...  
Keyword(s):  
Cell Density ◽  
Target Genes ◽  
High Cell Density ◽  
Critical Role ◽  
Susceptibility Gene ◽  
Mammary Tumors ◽  
Mrna Levels ◽  
High Cell ◽  
Wild Type ◽  
Induced Apoptosis

ABSTRACT Transcription factor ATF-2 is a nuclear target of stress-activated protein kinases, such as p38, which are activated by various extracellular stresses, including UV light. Here, we show that ATF-2 plays a critical role in hypoxia- and high-cell-density-induced apoptosis and the development of mammary tumors. Compared to wild-type cells, Atf-2 −/− mouse embryonic fibroblasts (MEFs) were more resistant to hypoxia- and anisomycin-induced apoptosis but remained equally susceptible to other stresses, including UV. Atf-2 −/− and Atf-2 +/− MEFs could not express a group of genes, such as Gadd45α, whose overexpression can induce apoptosis, in response to hypoxia. Atf-2 −/− MEFs also had a higher saturation density than wild-type cells and expressed lower levels of Maspin, the breast cancer tumor suppressor, which is also known to enhance cellular sensitivity to apoptotic stimuli. Atf-2 −/− MEFs underwent a lower degree of apoptosis at high cell density than wild-type cells. Atf-2 +/− mice were highly prone to mammary tumors that expressed reduced levels of Gadd45α and Maspin. The ATF-2 mRNA levels in human breast cancers were lower than those in normal breast tissue. Thus, ATF-2 acts as a tumor susceptibility gene of mammary tumors, at least partly, by activating a group of target genes, including Maspin and Gadd45α.

scholarly journals High Cell Density Cultivation of A Recombinant Bacillus Subtilis for Nattokinase Production

2020 ◽  
Author(s):  
Jianhua Zhang ◽  
Qing Cui ◽  
Bingjun Qian ◽  
Xiangjun Sun
Keyword(s):  
Enzyme Activity ◽  
Bacillus Subtilis ◽  
Cell Density ◽  
Large Scale ◽  
High Cell Density ◽  
Specific Activity ◽  
Feeding Strategy ◽  
Batch Process ◽  
High Cell ◽  
Fed Batch

Abstract Background: Nattokinase (NK), a fibrinolytic enzyme, can be produced by culturing recombinant Bacillus subtilis in Luria-Bertani broth in a shaking flask. For use as a nutraceutical, however, a large-scale preparation and a simple purification process are required.Results: The present study utilized a fed-batch process to cultivate a B. subtilis strain carrying a pHT01 plasmid with an NK-encoding gene (B. subtilis/pHT01-aprN1). For batch A (FB A), with a pH-stat two-stage fermentation strategy, we achieved an activity of 2910.5 ± 21.6 U mL-1 and a specific activity of 30.32 U ml-1 OD600-1. Then, we changed the strategy with a later induction and lower feeding rate to pursue higher cell density and thus higher enzyme activity, a 11.9-fold activity of 4521.8 ± 23.8 U mL-1 was acquired, however, the specific activity was lower than FB A. For the third batch, low-glycerol-level-maintain feeding strategy was followed, and finally, a NK activity of 7778 ±17.28 U mL-1 was obtained, according to our knowledge, it was the highest activity assayed by the fibrin plate method ever reported. Furthermore, fermentation supernatant was successively purified by ammonium sulfate precipitation and nickel column affinity chromatography with a total NK recovery rate of 65.2%.Conclusions: Our results indicate that there is a balance between the cell growth rate and NK expression when recombinant Bacillus subtilis is cultured with a fed-batch process. The equilibrium state can be attained by optimizing the induction and feeding strategy, and thus a high cell density and enzyme activity can be achieved.

scholarly journals Role of procollagen mRNA levels in controlling the rate of procollagen synthesis.

1983 ◽  
Vol 3 (2) ◽  
pp. 241-249 ◽  
Author(s):  
L B Rowe ◽  
R I Schwarz
Keyword(s):  
Cell Density ◽  
High Cell Density ◽  
Control Point ◽  
Relative Increase ◽  
Limiting Factor ◽  
Mrna Levels ◽  
Primary Control ◽  
High Cell ◽  
Mrna Induction ◽  
Tendon Cells

Two factors must be present for primary avian tendon cells to commit 50% of their total protein production to procollagen: ascorbate and high cell density. Scorbutic primary avian tendon cells at high cell density (greater than 4 X 10(4) cells per cm2) responded to the addition of ascorbate by a sixfold increase in the rate of procollagen synthesis. The kinetics were biphasic, showing a slow increase during the first 12 h followed by a more rapid rise to a maximum after 36 to 48 h. In contrast, after ascorbate addition, the level of accumulated cytoplasmic procollagen mRNA (alpha 2) showed a 12-h lag followed by a slow linear increase requiring 60 to 72 h to reach full induction. At all stages of the induction process, the relative increase in the rate of procollagen synthesis over the uninduced state exceeded the relative increase in the accumulation of procollagen mRNA. A similar delay in mRNA induction was observed when the cells were grown in an ascorbate-containing medium but the cell density was allowed to increase. In all cases, the rate of procollagen synthesis peaked approximately 24 h before the maximum accumulation of procollagen mRNA. The kinetics for the increase in procollagen synthesis are not, therefore, in agreement with the simple model that mRNA levels are the rate-limiting factor in the collagen pathway. We propose that the primary control point is at a later step. Further support for this idea comes from inhibitor studies, using alpha, alpha'-dipyridyl to block ascorbate action. In the presence of 0.3 mM alpha, alpha'-dipyridyl there was a specific two- to threefold decrease in procollagen production after 4 h, but this was unaccompanied by a drop in procollagen mRNA levels. Therefore, inhibitor studies give further support to the idea that primary action of ascorbate is to release a post-translational block.

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