Synthesis Of Β-galactosidase Research Articles

Freshwater microalgae Nannochloropsis limnetica for the production of β-galactosidase from whey powder

This study investigated the first-ever reported use of freshwater Nannochloropsis for the bioremediation of dairy processing side streams and co-generation of valuable products, such as β-galactosidase enzyme. In this study, N. limnetica was found to grow rapidly on both autoclaved and non-autoclaved whey-powder media (referred to dairy processing by-product or DPBP) without the need of salinity adjustment or nutrient additions, achieving a biomass concentration of 1.05–1.36 g L−1 after 8 days. The species secreted extracellular β-galactosidase (up to 40.84 ± 0.23 U L−1) in order to hydrolyse lactose in DPBP media into monosaccharides prior to absorption into biomass, demonstrating a mixotrophic pathway for lactose assimilation. The species was highly effective as a bioremediation agent, being able to remove > 80% of total nitrogen and phosphate in the DPBP medium within two days across all cultures. Population analysis using flow cytometry and multi-channel/multi-staining methods revealed that the culture grown on non-autoclaved medium contained a high initial bacterial load, comprising both contaminating bacteria in the medium and phycosphere bacteria associated with the microalgae. In both autoclaved and non-autoclaved DPBP media, Nannochloropsis cells were able to establish a stable microalgae–bacteria interaction, suppressing bacterial takeover and emerging as dominant population (53–80% of total cells) in the cultures. The extent of microalgal dominance, however, was less prominent in the non-autoclaved media. High initial bacterial loads in these cultures had mixed effects on microalgal performance, promoting β-galactosidase synthesis on the one hand while competing for nutrients and retarding microalgal growth on the other. These results alluded to the need of effective pre-treatment step to manage bacterial population in microalgal cultures on DPBP. Overall, N. limnetica cultures displayed competitive β-galactosidase productivity and propensity for efficient nutrient removal on DPBP medium, demonstrating their promising nature for use in the valorisation of dairy side streams.

Biodegradation and Metabolic Pathway of the Neonicotinoid Insecticide Thiamethoxam by Labrys portucalensis F11.

Thiamethoxam (TMX) is an effective neonicotinoid insecticide. However, its widespread use is detrimental to non-targeted organisms and water systems. This study investigates the biodegradation of this insecticide by Labrys portucalensis F11. After 30 days of incubation in mineral salt medium, L. portucalensis F11 was able to remove 41%, 35% and 100% of a supplied amount of TMX (10.8 mg L-1) provided as the sole carbon and nitrogen source, the sole carbon and sulfur source and as the sole carbon source, respectively. Periodic feeding with sodium acetate as the supplementary carbon source resulted in faster degradation of TMX (10.8 mg L-1); more than 90% was removed in 3 days. The detection and identification of biodegradation intermediates was performed by UPLC-QTOF/MS/MS. The chemical structure of 12 metabolites is proposed. Nitro reduction, oxadiazine ring cleavage and dechlorination are the main degradation pathways proposed. After biodegradation, toxicity was removed as indicated using Aliivibrio fischeri and by assessing the synthesis of an inducible β-galactosidase by an E. coli mutant (Toxi-Chromo test). L. portucalensis F11 was able to degrade TMX under different conditions and could be effective in bioremediation strategies.

Hydrothermal assisted eco-benign synthesis of novel β-galactosidase mediated Titanium dioxide nanoparticles (β-gal-TiO2 NPs): Ultra efficient nanocatalyst for methylene blue degradation, inactivation of bacteria, and stabilization of DPPH radicals

Water purification is a universal challenge and a global issue and most conventional approaches have several limitations. Therefore, new approaches must be introduced to address the challenges regarding water purification. In this contribution, a hydrothermal-assisted eco-benign protocol was followed for synthesizing titanium dioxide nanoparticles (β-gal-TiO2 NPs) using a novel β-galactosidase isolated from the seed extract of Melilots indica as stabilizing and reducing agent. technique Various physicochemical characterizations techniques examined the synthesis, crystalline structure, and surface morphology of the nanoparticles. The synthesized β-gal-TiO2 NPs were inspected for the photodecomposition of methylene blue (MB) and photoinhibition of bacteria. The results showed that β-gal-TiO2 NPs degraded 98% of MB under irradiation for only 60 min, exhibiting remarkable photocatalytic activity. Similarly, β-gal-TiO2 NPs displayed considerable antibacterial activity towards E. coli and S. aureus both in the presence and absence of light. The inhibition zones of irradiated β-gal-TiO2 NPs against E. coli and S. aureus were 23 mm and 19 mm, respectively significantly higher than in the dark. Moreover, β-gal-TiO2 NPs showed considerable antioxidant activity to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) effectively. Therefore, results demonstrated that eco-friendly synthesized β-gal-TiO2 NPs have great potential for bacterial pathogen remediation and MB degradation.

Effect of Saccharomyces cerevisiae on Probiotic Properties of Goat Milk Kefir

Goat milk kefir is a fermented milk product with kefir grain, as a probiotic agent and contains bioactive compounds. Kefir grains consist of bacteria and yeast. One of these yeasts are Saccharomyces cerevisiae, its role in the kefir system can be enhanced by restructure of kefir grain with increased Saccharomyces cerevisiae population. The objectives of this research were to analysis effect of the Saccharomyces cerevisiae concentration levels on kefir, as antibacterial against Escherichia coli, Salmonella typhi. Klebsiella pneumoniae, antioxidant activity, β-galactosidase synthesis. The research method was true experiment by Completely Randomized Design consist of 5 kind Saccharomyces cerevisiae concentration levels (S0: control; S1: 0.10%; S2: 0,25%; S3: 0.5% and S4: 1.0% v/w) of kefir grain. The results showed that kefir inhibited Escherichia coli, Salmonella typhi and Klebsiella pneumoniae, able to produce β-galactosidase and antioxidant activity. Kefir is relevant as food functional development.

Accurate Identification of Diverse N-acyl Homoserine Lactones in Marine Vibrio fluvialis by UHPLC-MS/MS.

Vibrio fluvialis is a marine opportunistic pathogen that frequently causes diseases in aquatic animals and humans. V. fluvialis can produce quorum sensing signaling molecules to coordinate cell density-dependent behavioral changes, including N-acyl homoserine lactone (AHL), which acts as a vital mediator of virulence-associated gene expression. Currently, several AHL molecules in V. fluvialis have been detected via biological and physicochemical methods, although different detection approaches have generated diverse AHL profiles. Here, we describe the AHL-producing bacterium, V. fluvialis BJ-1, which was isolated from marine sediments from the East China Sea. V. fluvialis BJ-1 could stimulate AHL-mediated β-galactosidase synthesis of the biosensor Agrobacterium tumefaciens NTL4 (pZLR4) but couldnot induce violacein production in the AHL reporter strain, Chromobacterium violaceum CV026. This bacterial isolate exhibited strong AHL-producing activity atlowcelldensity; however, the AHL activity declined when populationdensity remained at high levels. Analysis of the AHLs by Ultra-High-Performance Liquid Chromatography tandem Mass Spectrometry demonstrated that V. fluvialis BJ-1 produced five different AHL signaling molecules, including two linear chain AHL products (C8- and C10-HSL), and three β-carbon-oxidative AHL products (3-O-C8-, 3-O-C10- and 3-O-C12-HSL). Significantly, the present study is the first to accurately define the AHL profile of marine V. fluvialis. In future, the coupling of UHPLC to ESI-MS/MS is expected to be utilized for the accurate determination of AHL profiles in marine Vibrio.

An Improved Amperometric Lactose Biosensor Based on Enzyme Nanoparticles

A desolvation method was used for the synthesis of β-galactosidase (β-GAL) and glucose oxidase (GOD) nanoparticles (NPs) using ethanol. The enzyme nanoparticles (ENPs) were characterized by transmission electron microscopy (TEM) and Fourier transform Infra-red spectroscopy (FTIR) and were co-immobilized covalently onto surface of polycrystalline Au electrode. An improved amperometric lactose biosensor was constructed using ENPs (β-GALNPs/GODNPs) modified Au electrode as working electrode, Ag/AgCl as reference electrode and Pt wire as auxiliary electrode connected through potentiostat. Cyclic voltammetry (CV) and scanning electron microscope (SEM) were used for the study of working electrode before and after immobilization of ENPs. The biosensor showed maximum current at 0.25V within 5s, at pH 6.5 and 25oC. The limit of detection (LOD) was i.e. 1mg/ml and working range was 1-10 mg/mL. The analytical recovery of added lactose (5 and 10 mg/mL) was 94.73±0.5% and 96.4±0.9%, respectively. Coefficient of variation (CV) within and between batch were <3.0 and <4.0 respectively. The biosensor exhibited a good correlation (R2=0.91) between lactose level in milk as measured by standard (enzymatic colorimetric) method and present biosensor. The biosensor was employed for quantification of lactose in milk from human, cow, buffalo and goat. The working electrode lost 50% of its initial activity within 3 months, after its 120 uses, while being stored dry at 4ºC.
 Int. J. Appl. Sci. Biotechnol. Vol 10(1): 21-30.

Improved β-galactosidase Secretion in Fresh Water Fungi through Cultural Optimization

The wide range of applications of β-galactosidase has made it a candidate enzyme of study for improved yield. Most studies focused on expressing this enzyme in expressible vector(s) while sources of this enzyme are rarely investigated. Aquatic habitats harbor arrays of microorganisms that can participate in demineralization, nutrient restoration, circulation and hence confer significant properties on the habitats and significantly influence microbial interactions and metabolism. In view of this, aquatic fungal isolates were qualitatively and quantitatively screened for intracellular and extracellular β-galactosidase production. Four fungal isolates tagged SB01 and SC02 from stream identified as Aspergillus oryzae and Aspergillus niger and SC01 and SA01 from pond identified as A. oryzae and A. niger were studied. β-galactosidase gene was successfully amplified from the four isolates and their enzyme production levels were further studied. All the isolates showed considerably higher extracellular secretion than the intracellular synthesis on glucose-based medium. The production level was probed by xylose supplementation but surprisingly, the intracellular synthesis was higher than the extracellular secretion for all the isolates. In an attempt to boost the enzyme production, temperature was varied and 25 °C showed improved β-galactosidase synthesis and secretion than the 30 °C. This study revealed these filamentous fungi as candidates for β-galactosidase production and broaden the understanding of harnessing microorganisms from an aquatic reservoir for bio-production, and more importantly, revealed the intracellular accumulation of the enzyme as a bottleneck for industrial production.
 HIGHLIGHTS
 
 The wide range of applications of β-galactosidase has made it a candidate enzyme of study for improved yield
 Most studies focused on expressing this enzyme in expressible vector(s) while sources of this enzyme are rarely investigated
 Aquatic fungal isolates were qualitatively and quantitatively screened for intracellular and extracellular β-galactosidase production
 All the isolates showed considerable higher extracellular secretion than the intracellular synthesis on glucose-based medium, and vice-versa on xylose-supplemented medium, with the highest β-galactosidase synthesis and secretion efficiency occurring at 25 °C in both instances
 This study revealed these filamentous fungi as candidates for β-galactosidase production and broaden the understanding of harnessing microorganisms from an aquatic reservoir for bio-production
 
 GRAPHICAL ABSTRACT

Chlorella sorokiniana cultivation in cheese whey for β-galactosidase production

Biotechnological processes with microalgae with the aim to achieve high biomass yields must choose the appropriate nutrients and physicochemical parameters, taking into account the specific characteristics of each species to determine the basic needs for its growth. In the present study, the better growth condition of Chlorella sorokiniana IPR 7104 was optimized to reach the maximum beta-galactosidase production. The cheese whey concentration (%), temperature (˚C) and pH were factors investigated and a Box-Behnken Design (BBD) approach was implemented using Statistica 7.0 software. We observed that the cultivation condition to Chlorella sorokiniana IPR 7104 was the heterotrophic, which showed the major enzymatic activity, consequently a lower residual lactose content. Under heterotrophic conditions (without light) the β-galactosidase activity increased linearly until the 8th day. Biomass production grew linearly on the 12th day. The microalgae consumed 89.6% of lactose in 3 days, showing a high capacity to metabolize this disaccharide, through β-galactosidase synthesis. The maximum β-galactosidase production by Chlorella sorokiniana IPR 7104, in heterotrophic conditions and using cheese whey as carbon source, is obtained using the following conditions: 30°C temperature, concentration of ethanol at 20% and time of 4 min.

Nanosensors-Assisted Quantitative Analysis of Biochemical Processes in Droplets.

Here, we present a miniaturized lab-on-a-chip detecting system for an all-electric and label-free analysis of the emulsion droplets incorporating the nanoscopic silicon nanowires-based field-effect transistors (FETs). We specifically focus on the analysis of β-galactosidase e.g., activity, which is an important enzyme of the glycolysis metabolic pathway. Furthermore, the efficiency of the synthesis and action of β-galactosidase can be one of the markers for several diseases, e.g., cancer, hyper/hypoglycemia, cell senescence, or other disruptions in cell functioning. We measure the reaction and reaction kinetics-associated shift of the source-to-drain current Isd in the system, which is caused by the change of the ionic strength of the microenvironment. With these results, we demonstrate that the ion-sensitive FETs are able to sense the interior of the aqueous reactors; thus, the conjunction of miniature nanosensors and droplet-based microfluidic systems conceptually opens a new route toward a sensitive, optics-less analysis of biochemical processes.

I-Block: a simple Escherichia coli-based assay for studying sequence-specific DNA binding of proteins.

We have developed a simple method called I-Block assay, which can detect sequence-specific binding of proteins to DNA in Escherichia coli. The method works by detecting competition between the protein of interest and RNA polymerase for binding to overlapping target sites in a plasmid-borne lacI promoter variant. The assay utilizes two plasmids and an E. coli host strain, from which the gene of the Lac repressor (lacI) has been deleted. One of the plasmids carries the lacI gene with a unique NheI restriction site created in the lacI promoter. The potential recognition sequences of the tested protein are inserted into the NheI site. Introduction of the plasmids into the E. coliΔlacI host represses the constitutive β-galactosidase synthesis of the host bacterium. If the studied protein expressed from a compatible plasmid binds to its target site in the lacI promoter, it will interfere with lacI transcription and lead to increased β-galactosidase activity. The method was tested with two zinc finger proteins, with the lambda phage cI857 repressor, and with CRISPR-dCas9 targeted to the lacI promoter. The I-Block assay was shown to work with standard liquid cultures, with cultures grown in microplate and with colonies on X-gal indicator plates.

Perennial Prescience.

Perennial Prescience.

Regulation of phase variation in type I pili formation in Escherichia coli: Role of alkylresorcinols, microbial autoregulators

Formation of virulence-associated type I pili in Escherichia coli should be considered as one of the most efficient models for investigating the mechanisms of regulating the heterogeneity of populations of genetically identical microbial cells. The present work focused on the role of alkylhydroxybenzenes (AHBs), density-dependent intercellular regulators, in controlling phase variations in type I pili formation (fimbriogenesis). The tested AHB homologue was C12-AHB; a genetically constructed strain E. сoli dsp250 containing the fimA-lacZ hybrid operon was used. In this operon, the fimA gene encodes the main subunit of the pili protein, and its expression results in β-galactosidase synthesis; pili-forming cells, therefore, become blue on the medium with the Х-gal substrate. Expression of fimA depends on the inversion of the fimS region that is located upstream of it. If the inversion is on, pili formation takes place, if it is off, no pili are formed. An increase in C12-AHB concentration (within the 5 × 10–5–2 × 10–4 M range) in the exponential-phase culture of strain dsp250 causes a dose-dependent change in the dominant phenotype that is displayed by up to 98–99% of the cells. Cells with this phenotype form colonies with a blue center and white edges. Up to 60% of the cells with this phenotype assume a metastable state and up to 11% and 44% of them transition to the alternative phenotypes of pili-forming and pili-less cells, respectively. The influence of C12-AHB on off-switching, i.e. the formation of the avirulent phenotype, was observed irrespective of the growth conditions of strain dsp250. Addition of glucose to the LB medium (5 or 10 mg/mL) resulted in catabolic repression via regulation by the cAMP-CNR complex and predictably induced pili formation in 49 and 75% of the cells, respectively. Against this background, C12-AHB caused a dose-dependent decrease in the share of pili-forming cells to 33–61% and an increase in the share of pili-less cells to 32–61%. If glucose was added in excess (2.5, 5 or 10 mg/mL) to the diluted LB/2 medium, pili formation was completely repressed, while C12-AHB still induced the off inversion to the pili-less phenotype in up to 30% of the cells. The conclusion can be drawn that C12-AHB is not involved in the pathway of fimbriogenesis regulation via cAMP. Since C12-AHB functions as an extracellular alarmon (activating the rpoS regulon and the SOS response as shown earlier, see Golod et al., 2009), its mechanism of action apparently involves stress signal transduction. It induces the synthesis of global regulators RpoS and H-NS and of intracellular alarmon (p) ppGpp; these factors are responsible for the on → off inversion and the proliferation of pili-less cells.

Saccharomyces cerevisiae from Brazilian kefir-fermented milk: An invitro evaluation of probiotic properties.

The therapeutic use of probiotics for supporting the antibiotic action against gastrointestinal disorders is a current trend and emerging applications have gained popularity because of their support for various microbiological activities in digestive processes. Microorganisms isolated from kefir with great probiotic properties, in addition to high resistance to harsh environmental conditions, have been widely researched. Administration of probiotic yeasts offers a number of advantages, when compared to bacteria, because of particular characteristics as their larger cell size. In the present study, 28 strains of Saccharomyces cerevisiae were isolated, after invitro digestion of kefir-fermented milk, and identified by molecular based approaches. A screening was performed to determine important quality requirements for probiotics including: antagonistic and antioxidant activities, β-galactosidase synthesis, autoaggregation, surface hydrophobicity and adhesion to epithelial cells. The results showed strains: with antagonistic activity against microbial pathogens such as Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis; able to produce β-galactosidase; with antioxidant activity levels higher than 90%; with hydrophobicity activity and autoaggregation ability (evaluated by adhesion test, where all the strains presented adhesion to mice ileal epithelial cells). These findings are relevant and the strains are recommended for further invivo studies as well as for potential therapeutic applications.

Paper-based colorimetric biosensor for antibiotics inhibiting bacterial protein synthesis

Due to the presence of antibiotics in environmental water and their potential influence on the occurrence of antibiotic-resistant bacteria, development of a detection method suitable for the screening of environmental water for antibiotics is required. In this study, we developed a simple colorimetric paper-based biosensor based on a novel principle for the detection of antibiotics inhibiting bacterial protein synthesis, including aminoglycosides, tetracycline, chloramphenicol, and macrolides. This biosensor is based on the detection of a color change induced by β-galactosidase, which is synthesized on freeze-dried paper discs containing an invitro transcription/translation system. When a water sample without antibiotics is applied to the paper discs, β-galactosidase can be synthesized, and it hydrolyzes a colorimetric substrate, resulting in a color change from yellow to purple. By contrast, in the presence of antibiotics, the color change can be hampered due to an inhibition of β-galactosidase synthesis. We investigated the effect of the incubation temperature and pH of water samples and confirmed that the paper discs showed the color change to purple in the ranges of 15-37°C and pH 6-10. We observed concentration-dependent color variations of the paper discs by the naked eye and further estimated detection limits to be 0.5, 2.1, 0.8, and 6.1μg/mL for paromomycin, tetracycline, chloramphenicol, and erythromycin, respectively, using digitized pictures. The paper-based biosensor proved to detect 0.5μg/mL paromomycin, spiked in real environmental water samples, by the naked eye.

Mechanisms involved in xyloglucan catabolism by the cellulosome-producing bacterium Ruminiclostridium cellulolyticum.

Xyloglucan, a ubiquitous highly branched plant polysaccharide, was found to be rapidly degraded and metabolized by the cellulosome-producing bacterium Ruminiclostridium cellulolyticum. Our study shows that at least four cellulosomal enzymes displaying either endo- or exoxyloglucanase activities, achieve the extracellular degradation of xyloglucan into 4-glucosyl backbone xyloglucan oligosaccharides. The released oligosaccharides (composed of up to 9 monosaccharides) are subsequently imported by a highly specific ATP-binding cassette transporter (ABC-transporter), the expression of the corresponding genes being strongly induced by xyloglucan. This polysaccharide also triggers the synthesis of cytoplasmic β-galactosidase, α-xylosidase, and β-glucosidase that act sequentially to convert the imported oligosaccharides into galactose, xylose, glucose and unexpectedly cellobiose. Thus R. cellulolyticum has developed an energy-saving strategy to metabolize this hemicellulosic polysaccharide that relies on the action of the extracellular cellulosomes, a highly specialized ABC-transporter, and cytoplasmic enzymes acting in a specific order. This strategy appears to be widespread among cellulosome-producing mesophilic bacteria which display highly similar gene clusters encoding the cytosolic enzymes and the ABC-transporter.

Effect on β-galactosidase synthesis and burden on growth of osmotic stress in Escherichia coli.

Osmotic Shock is known to negatively affect growth rate along with an extended lag phase. The reduction in growth rate can be characterized as burden due to the osmotic stress. Studies have shown that production of unnecessary protein also burdens cellular growth. This has been demonstrated by growing Escherichia coli on glycerol in the presence of Isopropyl-β-D-1-thiogalactopyranoside (IPTG) to induce β-galactosidase synthesis which does not offer any benefit towards growth. The trade off between osmotic stress and burden on growth due to unnecessary gene expression has not been enumerated. The influence of osmotic stress on β-galactosidase synthesis and activity is not clearly understood. Here, we study the effect of salt concentration on β-galactosidase activity and burden on growth due to unnecessary gene expression in E.coli. We characterize the burden on growth in presence of varying concentrations of salt in the presence of IPTG using three strains, namely wild type, ∆lacI and ∆lacIlacZ mutant strains. We demonstrate that the salt concentrations, sensitively inhibits enzyme synthesis thereby influencing the burden on growth. In a wild type strain, addition of lactose into the medium demonstrated growth benefit at low salt concentration but not at higher concentrations. The extent of burden due to osmotic shock was higher in a lactose M9 medium than in a glycerol M9 medium. A linear relationship was observed between enzyme activity and burden on growth in various media types studied.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-748) contains supplementary material, which is available to authorized users.

Characterization of burden on growth due to the nutritional state of media and pre-induced gene expression

Studies have shown that the production of unnecessary proteins burdens the cellular growth mainly due to allocation of cellular resources to unnecessary protein synthesis, thereby limiting the resources available for growth. In the current study, we focus on the effect of pre-induction and nutritional status of the medium on the burden imposed on growth due to the synthesis of unnecessary protein. Escherichia coli cells with different history were grown in a glycerol media with and without IPTG to characterize the burden imposed due to the synthesis of β-galactosidase. Effect of pre-induced lac operon on growth and β-galactosidase expression on lactose milieu was also investigated. The study demonstrates that pre-induction has a strong influence on the extent of burden and is sustained in several generations. Further, the burden was much lower in a rich media relative to that observed in a minimal media.

Azelaic acid reduced senescence‐like phenotype in photo‐irradiated human dermal fibroblasts: possible implication of PPARγ

Azelaic acid (AzA) has been used for the treatment for inflammatory skin diseases, such as acne and rosacea. Interestingly, an improvement in skin texture has been observed after long-time treatment with AzA. We previously unrevealed that anti-inflammatory activity of AzA involves a specific activation of PPARγ, a nuclear receptor that plays a relevant role in inflammation and even in ageing processes. As rosacea has been considered as a photo-aggravated disease, we investigated the ability of AzA to counteract stress-induced premature cell senescence (SIPS). We employed a SIPS model based on single exposure of human dermal fibroblasts (HDFs) to UVA and 8-methoxypsoralen (PUVA), previously reported to activate a senescence-like phenotype, including long-term growth arrest, flattened morphology and increased synthesis of matrix metalloproteinases (MMPs) and senescence-associated β-galactosidase (SA-β-gal). We found that PUVA-treated HDFs grown in the presence of AzA maintained their morphology and reduced MMP-1 release and SA-β-galactosidase-positive cells. Moreover, AzA induced a reduction in ROS generation, an up-modulation of antioxidant enzymes and a decrease in cell membrane lipid damages in PUVA-treated HDFs. Further evidences of AzA anti-senescence effect were repression of p53 and p21, increase in type I pro-collagen and abrogation of the enhanced expression of growth factors, such as HGF and SCF. Interestingly, PUVA-SIPS showed a decreased activation of PPARγ and AzA counteracted this effect, suggesting that AzA effect involves PPARγ modulation. All together these data showed that AzA interferes with PUVA-induced senescence-like phenotype and its ability to activate PPAR-γ provides relevant insights into the anti-senescence mechanism.

Pap1p-dependent upregulation of thioredoxin 3 and thioredoxin reductase genes from the fission yeast under nitrosative stress

The thioredoxin system, consisting of thioredoxin, thioredoxin reductase, and NADPH, is involved in the response against a variety of stresses. The TRX3(+) and TrxR(+) genes encode thioredoxin 3 and thioredoxin reductase, respectively, in the fission yeast Schizosaccharomyces pombe . Their transcriptional regulations were studied using the lacZ fusion genes. Synthesis of β-galactosidase from the TRX3(+)-lacZ fusion gene was markedly enhanced by nitric-oxide-generating sodium nitroprusside in the Pap1p-positive cells but not in the Pap1p-negative cells. Similarly, synthesis of β-galactosidase from the TrxR(+)-lacZ fusion gene was upregulated by sodium nitroprusside in a Pap1p-dependent manner. Synthesis of β-galactosidase from the TRX3(+)-lacZ and TrxR(+)-lacZ fusion genes was also enhanced by S-nitrosoglutathione in the Pap1p-positive cells but not in the Pap1p-negative cells. In brief, the S. pombe genes encoding thioredoxin 3 and thioredoxin reductase are upregulated under nitrosative stress in a Pap1p-dependent manner.

Adenine-containing codons enhance protein synthesis by promoting mRNA binding to ribosomal 30S subunits provided that specific tRNAs are not exhausted

Adenines downstream of the initiation codon promote protein synthesis; however, some adenine-containing codons (AGA, AGG and AUA) at early positions inhibit protein synthesis when cognate tRNA is exhausted. It has also been reported, although not convincingly, the presence of adenines enhancing mRNA binding to the ribosome. To understand these apparent inconsistencies we analyzed the effect of these codons in mRNA-ribosome binding strength, mRNA stability, the production of peptidyl-tRNA (pep-tRNA) and protein synthesis. Constructs harboring lacZ derivatives were obtained by site directed mutagenesis where tandems of GGG, AGG, AGA, ATA and AAA codons were inserted at codon positions 2–3 and 3–4. Codons containing more adenines, irrespective of being common or rare, (AAA, ATA and AGA) promoted a higher synthesis of β-galactosidase (β-gal) in comparison with those rich in guanines (GGG and AGG) in a wild type transcription-translation system. Full-length mRNAs were also detected when the adenine-rich constructs were expressed in wild type cells. Under conditions where the pool of tRNAs is readily exhausted (pep-tRNA hydrolase defective cells), the adenine-rich lacZ derivatives caused a stronger and general inhibition of protein synthesis and cell growth. With the exception of the ATA lacZ derivative, only plasmid constructs containing hungry codons generated pep-tRNA (AGA and to a lesser extent AGG) in Pth defective cells. Codons containing more adenines clearly promoted lacZ mRNA binding to 30S subunit. The GGG lacZ mRNA showed a moderate increase in binding when mRNA secondary structures were disrupted by heating mRNAs before the binding assay which agrees with the lacZ mRNA secondary structures predicted with MFOLD. Altogether, these results indicate that mRNA binding to ribosome plays a major role in the enhancement of translation by adenine-rich codons irrespective of codon usage. This effect is naturally expressed in wild type systems and depends on adenine content, in contrast to the inhibition caused after over-expressing the lacZ derivatives containing rare codons in Pth defective cells.