NADH- vs NADPH-coupled reduction of 5-hydroxymethyl furfural (HMF) and its implications on product distribution in Saccharomyces cerevisiae

Saccharomyces cerevisiae alcohol dehydrogenases responsible for NADH-, and NADPH-specific reduction of the furaldehydes 5-hydroxymethyl-furfural (HMF) and furfural have previously been identified. In the present study, strains overexpressing the corresponding genes (mut-ADH1 and ADH6), together with a control strain, were compared in defined medium for anaerobic fermentation of glucose in the presence and absence of HMF. All strains showed a similar fermentation pattern in the absence of HMF. In the presence of HMF, the strain overexpressing ADH6 showed the highest HMF reduction rate and the highest specific ethanol productivity, followed by the strain overexpressing mut-ADH1. This correlated with in vitro HMF reduction capacity observed in the ADH6 overexpressing strain. Acetate and glycerol yields per biomass increased considerably in the ADH6 strain. In the other two strains, only the overall acetate yield per biomass was affected. When compared in batch fermentation of spruce hydrolysate, strains overexpressing ADH6 and mut-ADH1 had five times higher HMF uptake rate than the control strain and improved specific ethanol productivity. Overall, our results demonstrate that (1) the cofactor usage in the HMF reduction affects the product distribution, and (2) increased HMF reduction activity results in increased specific ethanol productivity in defined mineral medium and in spruce hydrolysate.     

Regulation of UDP-glucose pyrophosphorylase isozyme UGP5 associated with cold-sweetening resistance in potatoes

The regulation of UDP-Glc pyrophosphorylase (UGPase) isozyme, UGP5, was investigated in potato tuber. The cDNA for UGP5 was cloned into the bacterial expression vector pET21d and recombinant (RC) enzyme was expressed in E. coli (BL21 star cells). The RC-UGP5 isozyme was purified to near homogeneity using salt precipitation, hydrophobic interaction, and anion-exchange column chromatography. Kinetic analysis revealed that in the synthesis direction, K(m) values for Glc-1-P (0.83mM) and UTP (0.22mM) were similar to those observed previously with the mother tuber (MT)-UGP5. In the pyrophosphorolysis direction, the K(m) values for UDP-Glc (0.68mM) and PPi (0.56mM) were slightly higher than those observed previously. Maximum reaction velocities (V(max)) for RC-UGP5 were also elevated. Since the molecular mass, charge, and amino acid sequence of the MT- and RC-UGP5 isozymes were identical, it was assumed that altered kinetic constants may be due to an improper folding of RC-UGP5 polypeptide. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and proteomic analysis demonstrated that the UGP5 isozyme was a single polypeptide with a calculated molecular mass of 51.8kDa consisting of 477 amino acids. Native PAGE and kinetic analysis revealed that this polypeptide was monomeric in nature. Immunoblotting with specific antibodies and LC-MS/MS data indicated that UGP5 did not require any post-translational modification (e.g., phosphorylation, O-glycosylation, oligomerization/de-oligomerization, or the presence of the regulatory 14-3-3 proteins) for its regulation. Additionally, the two closely associated isozymes UGP5 and UGP6 in the cv. Snowden are likely the result of allelic differences of UGPase at a single locus.     

RIP2 regulates growth and differentiation of normal myoblasts and of rhabdomyosarcoma cells

RIP2 is an important regulator of myoblast proliferation and differentiation. We have previously demonstrated that in the myoblast cell line C2C12 and in primary myoblasts, downregulation of rip2 gene expression is a prerequisite for differentiation. To further study the role of rip genes in myogenesis, we compared expression patterns of rip1–4 in two myoblast cell lines, C2C12 and C2F3, after the induction of differentiation. These two cell lines are derived from the same clonal origin, but differ with respect to their differentiation behaviour: specifically, the differentiation process is slower and more incomplete in C2F3 cells. When analyzing cells up to 4 days after the induction of differentiation, we found no downregulation of rip2 gene expression in C2F3 cells, which might be linked to the low differentiation potential of these cells. In addition, in contrast to C2C12 cells, the rip3 gene was not expressed in C2F3 cells. To further study the role of rip genes in the regulation of myoblast growth and differentiation, we analyzed expression patterns of rip1–4 in rhabdomyosarcoma cell lines. We found that in these cells, rip2 expression was not downregulated after the induction of differentiation. Furthermore, in contrast to normal myoblasts, they did not express the rip3 and rip4 genes. Thus, we focused on the functional role of RIP2 in rhabdomyosarcoma cells. Inhibition of rip2 gene expression in C2C12 and in rhabdomyosarcoma cells using specific siRNAs led to decreased proliferation and promoted the differentiation process of these cells. These data indicate that differential expression of rip genes can be associated with abnormal growth and differentiation behaviour of skeletal myoblasts.     

The role of protein–solvent hydrogen bond dynamics in the structural relaxation of a protein in glycerol versus water

We used MD simulations to investigate the dependence of the dynamics of a soluble protein, RNase A, on temperature and solvent environment. Consistent with neutron scattering data, the simulations predict that the protein undergoes a dynamical transition in both glycerol and aqueous solutions that is absent in the dry protein. The temperature of the transition is higher, while the rate of increase with temperature of the amplitudes of motion on the 100 ps timescale is lower, in glycerol versus water. Analysis of the dynamics of hydrogen bonds revealed that the protein dynamical transition is connected to the relaxation of the protein-solvent hydrogen bond network, which, in turn, is associated with solvent translational diffusion. Thus, it appears that the role of solvent dynamics in affecting the protein dynamical transition is qualitatively similar in water and glycerol.     

Brown adipose tissue specific lack of uncoupling protein 3 is associated with impaired cold tolerance and reduced transcript levels of metabolic genes

Uncoupling protein 3 (Ucp3) is located within the mitochondrial inner membrane of brown adipose tissue and skeletal muscle. It is thought to be implicated in lipid metabolism and defense against reactive oxygen species. We previously reported on a mutation in our breeding colony of Djungarian hamsters (Phodopus sungorus) that leads to brown adipose tissue specific lack of Ucp3 expression. In this study we compared wildtype with mutant hamsters on a broad genetic background. Hamsters lacking Ucp3 in brown adipose tissue displayed a reduced cold tolerance due to impaired nonshivering thermogenesis. This phenotype is associated with a global decrease in expression of metabolic genes but not of uncoupling protein 1. These data implicate that Ucp3 is necessary to sustain high metabolic rates in brown adipose tissue.     

Establishment of adventitious root co-culture of Ginseng and Echinacea for the production of secondary metabolites

We have successfully established the co-culture of ginseng (Panax ginseng C.A. Meyer) and echinacea [Echiancea purpurea (L.) Moench.] adventitious roots for the production secondary metabolites. Adventitious roots of ginseng and echinacea were cultured in different proportions (5 g L⁻¹; 4:1, 3:2 and 2:1 ginseng and echinacea, respectively) in 5-L capacity airlift bioreactors containing 4 L Murashige and Skoog medium supplemented with 25 μM indole-3-butyric acid and 50 g sucrose L⁻¹ and maintained at 25°C in the dark for 40 days. Results showed the negative effect of echinacea adventitious roots on the growth of ginseng roots, however, by limiting the inoculum density of echinacea, it was possible to establish the co-cultures. To enhance the accumulation of secondary metabolites, co-cultures were treated with 200 μM methyl jasmonate after 30 days of culture initiation. Methyl jasmonate elicitation promoted the accumulation of ginsenosides in the co-cultures. It was possible to produce ginsenosides and caffeic acid derivatives in higher amounts by establishing co-cultures with higher inoculum proportion of ginseng to echinacea (4:1 and 3:2) followed by elicitation treatment. This work demonstrates the effectiveness of interspecies adventitious root co-cultures for the production of plant secondary metabolites.     

Ultrafiltration-based techniques for rapid and simultaneous concentration of multiple microbe classes from 100-L tap water samples

This study focused on ultrafiltration as a technique for simultaneously concentrating and recovering viruses, bacteria and parasites in 100-L drinking water samples. A chemical dispersant, sodium polyphosphate, and Tween 80 were used to increase microbial recovery efficiencies. Secondary concentration was performed to reduce sample volumes to 3-5 mL for analysis using tissue culture, microscopy, and real-time PCR and RT-PCR. At seeding levels of 100-1000 (CFU, PFU, oocysts, or particles), a "high-flux" ultrafiltration procedure was found to achieve mean recoveries of 51-94% of simultaneously seeded MS2 bacteriophage, echovirus 1, Salmonella enterica subsp. enterica serovar Typhimurium, Bacillus atrophaeus subsp. globigii endospores, Cryptosporidium parvum oocysts, and 4.5-mum microspheres. When 4-7% of the final sample concentrate volume was assayed using real-time PCR and RT-PCR, overall method sensitivities were <100 C. parvum oocysts, <240 PFU echovirus 1, <100 CFU Salmonella and approximately 160 CFU B. atrophaeus spores in 100-L drinking water samples. The "high-flux" ultrafiltration procedure required approximately 2 h, including time required for backflushing. Secondary concentration procedures required an additional 1-3 h, while nucleic acid extraction and real-time PCR procedures required an additional 2-2.5 h. Thus, this study demonstrated that efficient recovery and sensitive detection of diverse microbes in 100-L drinking water samples could be achieved within 5-8 h using ultrafiltration, rapid secondary processing techniques, and real-time PCR.