Previous studies have focused on the effect of individual stressors on hsp gene expression in eukaryotic organisms. In the present study, I examined the effect of concurrent low doses of sodium arsenite and mild heat shock temperatures on the expression of hsp30 and hsp70 genes in Xenopus laevis A6 kidney epithelial cells. Northern hybridization and western blot analysis revealed that exposure of A6 cells to 1-10 μM sodium arsenite at a mild heat shock temperature of 30˚C enhanced hsp30 and hsp70 gene expression to a much greater extent than found with either stress individually. In cells treated simultaneously with 10 μM sodium arsenite and different heat shock temperatures, enhanced accumulation of HSP30 and HSP70 protein was first detected at 26˚C with larger responses at 28 and 30 ˚C. HSF1 activity was involved in combined stress-induced hsp gene expression since the HSF1 activation inhibitor, KNK437, inhibited HSP30 and HSP70 accumulation. Immunocytochemical analysis revealed that HSP30 was present in a granular pattern primarily in the cytoplasm in cells treated simultaneously with both stresses. Finally, prior exposure of A6 cells to concurrent sodium arsenite (10 μM ) and heat shock (30 ˚C) treatment conferred thermotolerance since it protected them against a subsequent thermal challenge at 37 ˚C. Acquired thermotolerance was not observed with cells treated with the two mild stresses individually. It is likely that the enhanced accumulation of HSPs under these conditions permits the organism to cope with multiple environmental stresses encountered in their natural aquatic habitat. Previous studies have shown that inhibiting the activity of the proteasome also leads to the accumulation of damaged or unfolded proteins within the cell. In the second phase of this study, I report that inhibition of proteasome activity by the inhibitors carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132) and lactacystin induced the accumulation of HSP30 and HSP70 as well as their respective mRNAs. The accumulation of HSP30 and HSP70 in A6 cells recovering from MG132 exposure was still relatively high 24 h after treatment and it decreased substantially after 48 h. Exposing A6 cells to simultaneous MG132 and mild heat shock enhanced the accumulation of HSP30 and HSP70 to a much greater extent than with each stressor alone. HSP30 localization in A6 cells was primarily in the cytoplasm as revealed by immunocytochemistry. In some A6 cells treated with higher concentrations of MG132 and lactacystin, HSP30 was also found to localize in relatively large cytoplasmic foci. In some MG132-treated cells, HSP30 staining was substantially depleted in the cytoplasmic regions surrounding these foci. The activation of HSF1 may be involved in MG132-induced hsp gene expression in A6 cells since KNK437 inhibited the accumulation of HSP30 and HSP70. Lastly, MG132 treatment also conferred a state of thermotolerance in A6 cells such that they were able to survive a subsequent thermal challenge. Analysis of this phenomenon is important given the fact that impaired proteasomal activity has been suggested as an explanation for some of the late-onset neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease.

Previous studies have focused on the effect of individual stressors on hsp gene expression in eukaryotic organisms. In the present study, I examined the effect of concurrent low doses of sodium arsenite and mild heat shock temperatures on the expression of hsp30 and hsp70 genes in Xenopus laevis A6 kidney epithelial cells. Northern hybridization and western blot analysis revealed that exposure of A6 cells to 1-10 μM sodium arsenite at a mild heat shock temperature of 30˚C enhanced hsp30 and hsp70 gene expression to a much greater extent than found with either stress individually. In cells treated simultaneously with 10 μM sodium arsenite and different heat shock temperatures, enhanced accumulation of HSP30 and HSP70 protein was first detected at 26˚C with larger responses at 28 and 30 ˚C. HSF1 activity was involved in combined stress-induced hsp gene expression since the HSF1 activation inhibitor, KNK437, inhibited HSP30 and HSP70 accumulation. Immunocytochemical analysis revealed that HSP30 was present in a granular pattern primarily in the cytoplasm in cells treated simultaneously with both stresses. Finally, prior exposure of A6 cells to concurrent sodium arsenite (10 μM ) and heat shock (30 ˚C) treatment conferred thermotolerance since it protected them against a subsequent thermal challenge at 37 ˚C. Acquired thermotolerance was not observed with cells treated with the two mild stresses individually. It is likely that the enhanced accumulation of HSPs under these conditions permits the organism to cope with multiple environmental stresses encountered in their natural aquatic habitat. Previous studies have shown that inhibiting the activity of the proteasome also leads to the accumulation of damaged or unfolded proteins within the cell. In the second phase of this study, I report that inhibition of proteasome activity by the inhibitors carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132) and lactacystin induced the accumulation of HSP30 and HSP70 as well as their respective mRNAs. The accumulation of HSP30 and HSP70 in A6 cells recovering from MG132 exposure was still relatively high 24 h after treatment and it decreased substantially after 48 h. Exposing A6 cells to simultaneous MG132 and mild heat shock enhanced the accumulation of HSP30 and HSP70 to a much greater extent than with each stressor alone. HSP30 localization in A6 cells was primarily in the cytoplasm as revealed by immunocytochemistry. In some A6 cells treated with higher concentrations of MG132 and lactacystin, HSP30 was also found to localize in relatively large cytoplasmic foci. In some MG132-treated cells, HSP30 staining was substantially depleted in the cytoplasmic regions surrounding these foci. The activation of HSF1 may be involved in MG132-induced hsp gene expression in A6 cells since KNK437 inhibited the accumulation of HSP30 and HSP70. Lastly, MG132 treatment also conferred a state of thermotolerance in A6 cells such that they were able to survive a subsequent thermal challenge. Analysis of this phenomenon is important given the fact that impaired proteasomal activity has been suggested as an explanation for some of the late-onset neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease.

The loss of efficiency and performance of bioprocesses on scale-up is well known, but not fully understood. This work addresses this problem, by studying the effect of some fermentation gradients (pH, glucose and oxygen) at a larger scale in a bench-scale two compartment reactor (PFR + STR) using the cadaverine-producing recombinant bacterium, Corynebacterium glutamicum DM1945 Δact3 Ptuf-ldcC_OPT. The initial scale down strategy increased the magnitude of these gradients by only increasing the mean cell residence time in the plug flow reactor (τ_PFR). The cell growth and product related rate constants were compared as the τ_PFR was increased; differences were significant in some cases, but only up to 2 min residence time. For example, losses in cadaverine productivity when compared to the control fed-batch fermentation on average for the τ_PFR of 1 min, 2 min and 5 min were 25 %, 42 % and 46 % respectively. This indicated that the increasing the τ_PFR alone does not necessarily increase the magnitude of fermentation gradients. The new scale-down strategy developed here, increased the magnitude of fermentation gradients by not only increasing the τ_PFR, but also considering the mean frequency at which the bacterial cells entered the PFR section (f_m). The f_m was kept constant by reducing the broth volume in the STR. Hence, the bacterial cells also spent shorter times in the well mixed STR, as the τ_PFR was increased (hypothesised as giving the bacterial cells less time to recover the non-ideal PFR section of the SDR). On adoption of this strategy cadaverine productivity decreases for the τ_PFR of 1 min, 2 min and 5 min were 25 %, 32 % and 53 % respectively. Thus, highlighting that loss in performance is most likely to occur as the magnitude of heterogeneity within the fermentation environment increases. However, Corynebacterium glutamicum DM1945 Δact3 Ptuf-ldcC_OPT did show some resilience in its biomass productivity. It was only marginally affected in the harshest of conditions simulated here.