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111.

Background

Glycerol is the major by-product accounting for up to 5% of the carbon in Saccharomyces cerevisiae ethanolic fermentation. Decreasing glycerol formation may redirect part of the carbon toward ethanol production. However, abolishment of glycerol formation strongly affects yeast's robustness towards different types of stress occurring in an industrial process. In order to assess whether glycerol production can be reduced to a certain extent without jeopardising growth and stress tolerance, the yeast's capacity to synthesize glycerol was adjusted by fine-tuning the activity of the rate-controlling enzyme glycerol 3-phosphate dehydrogenase (GPDH). Two engineered strains whose specific GPDH activity was significantly reduced by two different degrees were comprehensively characterized in a previously developed Very High Ethanol Performance (VHEP) fed-batch process.

Results

The prototrophic strain CEN.PK113-7D was chosen for decreasing glycerol formation capacity. The fine-tuned reduction of specific GPDH activity was achieved by replacing the native GPD1 promoter in the yeast genome by previously generated well-characterized TEF promoter mutant versions in a gpd2 Δ background. Two TEF promoter mutant versions were selected for this study, resulting in a residual GPDH activity of 55 and 6%, respectively. The corresponding strains were referred to here as TEFmut7 and TEFmut2. The genetic modifications were accompanied to a strong reduction in glycerol yield on glucose; the level of reduction compared to the wild-type was 61% in TEFmut7 and 88% in TEFmut2. The overall ethanol production yield on glucose was improved from 0.43 g g-1 in the wild type to 0.44 g g-1 measured in TEFmut7 and 0.45 g g-1 in TEFmut2. Although maximal growth rate in the engineered strains was reduced by 20 and 30%, for TEFmut7 and TEFmut2 respectively, strains' ethanol stress robustness was hardly affected; i.e. values for final ethanol concentration (117 ± 4 g L-1), growth-inhibiting ethanol concentration (87 ± 3 g L-1) and volumetric ethanol productivity (2.1 ± 0.15 g l-1 h-1) measured in wild-type remained virtually unchanged in the engineered strains.

Conclusions

This work demonstrates the power of fine-tuned pathway engineering, particularly when a compromise has to be found between high product yield on one hand and acceptable growth, productivity and stress resistance on the other hand. Under the conditions used in this study (VHEP fed-batch), the two strains with "fine-tuned" GPD1 expression in a gpd2 Δ background showed slightly better ethanol yield improvement than previously achieved with the single deletion strains gpd1 Δ or gpd2 Δ. Although glycerol reduction is known to be even higher in a gpd1 Δ gpd2 Δ double deletion strain, our strains could much better cope with process stress as reflected by better growth and viability.  相似文献   
112.
The vacuolating toxin VacA, released by Helicobacter pylori, is an important virulence factor in the pathogenesis of gastritis and gastroduodenal ulcers. VacA contains two subunits: The p58 subunit mediates entry into target cells, and the p34 subunit mediates targeting to mitochondria and is essential for toxicity. In this study we found that targeting to mitochondria is dependent on a unique signal sequence of 32 uncharged amino acid residues at the p34 N-terminus. Mitochondrial import of p34 is mediated by the import receptor Tom20 and the import channel of the outer membrane TOM complex, leading to insertion of p34 into the mitochondrial inner membrane. p34 assembles in homo-hexamers of extraordinary high stability. CD spectra of the purified protein indicate a content of >40% β-strands, similar to pore-forming β-barrel proteins. p34 forms an anion channel with a conductivity of about 12 pS in 1.5 M KCl buffer. Oligomerization and channel formation are independent both of the 32 uncharged N-terminal residues and of the p58 subunit of the toxin. The conductivity is efficiently blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), a reagent known to inhibit VacA-mediated apoptosis. We conclude that p34 essentially acts as a small pore-forming toxin, targeted to the mitochondrial inner membrane by a special hydrophobic N-terminal signal.  相似文献   
113.
Natural vitamin E consists of four different tocopherol and four different tocotrienol homologues (alpha,beta, gamma, delta) that all have antioxidant activity. However, recent data indicate that the different vitamin E homologues also have biological activity unrelated to their antioxidant activity. In this review, we discuss the anti-inflammatory properties of the two major forms of vitamin E, alpha-tocopherol (alphaT) and gamma-tocopherol (gammaT), and discuss the potential molecular mechanisms involved in these effects. While both tocopherols exhibit anti-inflammatory activity in vitro and in vivo, supplementation with mixed (gammaT-enriched) tocopherols seems to be more potent than supplementation with alphaT alone. This may explain the mostly negative outcomes of the recent large-scale interventional chronic disease prevention trials with alphaT only and thus warrants further investigation.  相似文献   
114.
The Galanthus nivalis agglutinin (GNA) is synthesized as a preproprotein. To corroborate the role of the different targeting peptides in the topogenesis of GNA and related proteins, different constructs were made whereby both the complete original GNA gene and different truncated sequences were coupled to the enhanced green fluorescent protein (EGFP). In addition, a GNA ortholog from rice that lacks the signal peptide and C-terminal propeptide sequence was fused to EGFP. These fusion constructs were expressed in tobacco BY-2 cells and their localization analyzed by confocal fluorescence microscopy. We observed that the processed preproprotein of GNA was directed towards the vacuolar compartment, whereas both the truncated forms of GNA corresponding to the mature lectin polypeptide and the rice ortholog of GNA were located in the nucleus and the cytoplasm. It can be concluded, therefore, that removal of the C-terminal propeptide and the signal peptide is sufficient to change the subcellular targeting of a normally vacuolar protein to the nuclear/cytoplasmic compartment of the BY-2 cells. These findings support the proposed hypothesis that cytoplasmic/nuclear GNA-like proteins and their vacuolar homologs are evolutionarily related and that the classical GNA-related lectins might have evolved from cytoplasmic orthologs through an evolutionary event involving the insertion of a signal peptide and a C-terminal propeptide.  相似文献   
115.
Tripeptidyl peptidase II (TPPII) is an oligopeptidase forming giant complexes in the cytosol that have high exo-, but also, endoproteolytic activity. Immunohistochemically, the complexes appear as distinct foci in the cytosol. In part controversial biochemical and functional studies have suggested that TPPII contributes, on the one hand, positively to Ag processing by generating epitope carboxyl termini or by trimming epitope precursors, and, on the other, negatively by destroying potentially antigenic peptides. To clarify which of these roles is predominant, we generated and analyzed TPPII-deficient mice. Cell surface levels of MHC class I peptide complexes tended to be increased on most cell types of these mice. Although presentation of three individual epitopes derived from lymphocytic choriomeningitis virus was not elevated on TPPII-/- cells, that of the immunodominant OVA epitope SIINFEKL was significantly enhanced. Consistent with this, degradation of a synthetic peptide corresponding to the OVA epitope and of another corresponding to a precursor thereof, both being proteasomally generated OVA fragments, was delayed in TPPII-deficient cytosolic extracts. In addition, dendritic cell cross-presentation of phagocytosed OVA and of OVA internalized as an immune complex was increased to about the same level as direct presentation of the Ag. The data suggest a moderate, predominantly destructive role of TPPII in class I Ag processing, in line with our finding that TPPII is not induced by IFN-gamma, which up-regulates numerous, predominantly constructive components of the Ag processing and presentation machinery.  相似文献   
116.
Male mating tactics can vary according to the potential for scramble or contest competition but also as a consequence of individual characteristics, such as body condition and previous experience. The influence of experience, i.e., residency, on male recapture rates and reproductive success was studied in a population of free-living grey mouse lemurs. Long-term capture data from 320 individuals revealed that both sexes had very low recapture probabilities within their first year in the study population, but recapture rates declined less sharply during the following years. Capture results and telemetric analyses on 12 focal males revealed that resident males had larger body mass and larger home ranges than new males. Home range size correlated with the number of accessible females, indicating that resident males had higher probabilities to meet mates than new males. The reproductive success of 132 candidate fathers, representing both resident and new males, was determined by means of molecular genotyping. Paternity determination was successful in 38 cases (success rate: 19%). Sixteen resident males and seventeen new males sired offspring. However, in relation to the number of candidate fathers being present in the mating season, resident males were twice as likely to reproduce successfully as new males. These findings suggest experience-dependent reproductive tactics that most likely correspond to a differential spatial knowledge of resources, mates and potential threats. The results generally agree with the predictions made for a scramble competition regime and demonstrate substantial behavioral plasticity in a nocturnal primate species with a dispersed multi-male/multi-female mating system.  相似文献   
117.
The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) cascade plays a central role in intracellular signaling by many extracellular stimuli. One target of the ERK cascade is peroxisome proliferator-activated receptor gamma (PPARgamma), a nuclear receptor that promotes differentiation and apoptosis. It was previously demonstrated that PPARgamma activity is attenuated upon mitogenic stimulation due to phosphorylation of its Ser84 by ERKs. Here we show that stimulation by tetradecanoyl phorbol acetate (TPA) attenuates PPARgamma's activity in a MEK-dependent manner, even when Ser84 is mutated to Ala. To elucidate the mechanism of attenuation, we found that PPARgamma directly interacts with MEKs, which are the activators of ERKs, but not with ERKs themselves, both in vivo and in vitro. This interaction is facilitated by MEKs' phosphorylation and is mediated by the basic D domain of MEK1 and the AF2 domain of PPARgamma. Immunofluorescence microscopy and subcellular fractionation revealed that MEK1 exports PPARgamma from the nucleus, and this finding was supported by small interfering RNA knockdown of MEK1 and use of a cell-permeable interaction-blocking peptide, which prevented TPA-induced export of PPARgamma from the nucleus. Thus, we show here a novel mode of downregulation of PPARgamma by its MEK-dependent redistribution from the nucleus to the cytosol. This unanticipated role for the stimulation-induced nuclear shuttling of MEKs shows that MEKs can regulate additional signaling components besides the ERK cascade.  相似文献   
118.
The structural maintenance of chromosome (Smc) protein is highly conserved and involved in chromosome compaction, cohesion, and other DNA-related processes. In Bacillus subtilis, smc null mutations cause defects in DNA supercoiling, chromosome compaction, and chromosome partitioning. We investigated the effects of smc mutations on global gene expression in B. subtilis using DNA microarrays. We found that an smc null mutation caused partial induction of the SOS response, including induction of the defective prophage PBSX. Analysis of SOS and phage gene expression in single cells indicated that approximately 1% of smc mutants have fully induced SOS and PBSX gene expression while the other 99% of cells appear to have little or no expression. We found that induction of PBSX was not responsible for the chromosome partitioning or compaction defects of smc mutants. Similar inductions of the SOS response and PBSX were observed in cells depleted of topoisomerase I, an enzyme that relaxes negatively supercoiled DNA.  相似文献   
119.
Empirical research on human and non-human primates suggests that communication sounds express the intensity of an emotional state of a signaller. In the present study, we have examined communication sounds during induced social interactions of a monogamous mammal, the tree shrew. To signal their unwillingness to mate, female tree shrews show defensive threat displays towards unfamiliar males paralleled by acoustically variable squeaks. We assumed that the distance between interacting partners as well as the behavior of the male towards the female indicates the intensity of perceived social threat and thereby the arousal state of a female. To explore this hypothesis we analyzed dynamic changes in communication sounds uttered during induced social interactions between a female and an unfamiliar male. Detailed videographic and sound analyzes revealed that the arousal state predicted variations in communication sound structure reliably. Both, a decrease of distance and a male approaching the female led to an increase in fundamental frequency and repetition rate of syllables. These findings support comparable results in human and non-human primates and suggest that common coding rules in communication sounds govern acoustic conflict regulation in mammals.  相似文献   
120.
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