Osmolytes Betaine Research Articles

The organic osmolyte betaine effectively counteracts the deleterious effect of diclofenac on protein structure and stability

Understanding the physicochemical properties of pharmacy and the concept of pharmacophore is essential to devise guidelines for the synthesis of target-oriented drugs. In the present study, thermal denaturation, circular dichroism, intrinsic fluorescence, and docking were employed for studying the effects of the non-steroidal anti-inflammatory drug, diclofenac sodium, on the stability and structure of bovine pancreatic ribonuclease (RNase A) taken as a model of an enzyme. Since drug-protein interaction is an important pharmacokinetic parameter of a drug, it was deemed significant to study the effects of diclofenac on protein structure and stability. When the thermodynamic parameters were assessed, it was shown that RNase-A was destabilized in the presence of diclofenac sodium in terms of Tm and ΔGD°, while the addition of osmolytes stabilized the protein. In the presence of diclofenac sodium and osmolyte, there was a change in a tertiary structure of the protein but not in its secondary structure. Spectral results revealed a decrease in fluorescence intensity confirming fluorescence quenching of RNase-A by diclofenac sodium and osmolytes. Molecular docking analysis suggested that hydrogen bonds and Van der Waals are involved in the binding of RNase-A to diclofenac sodium. The RNAse-A activity is decreased in the presence of diclofenac sodium while osmolyte can restore the RNase-A activity and the indices kcat increase and Km values decrease. This study presents new combination of drug therapy comprising diclofenac sodium and osmolytes to reduce the risk of adverse effects of diclofenac sodium.

Exploiting the mediating role of the metabolome to unravel transcript-to-phenotype associations

Despite the success of genome-wide association studies (GWASs) in identifying genetic variants associated with complex traits, understanding the mechanisms behind these statistical associations remains challenging. Several methods that integrate methylation, gene expression, and protein quantitative trait loci (QTLs) with GWAS data to determine their causal role in the path from genotype to phenotype have been proposed. Here, we developed and applied a multi-omics Mendelian randomization (MR) framework to study how metabolites mediate the effect of gene expression on complex traits. We identified 216 transcript-metabolite-trait causal triplets involving 26 medically relevant phenotypes. Among these associations, 58% were missed by classical transcriptome-wide MR, which only uses gene expression and GWAS data. This allowed the identification of biologically relevant pathways, such as between ANKH and calcium levels mediated by citrate levels and SLC6A12 and serum creatinine through modulation of the levels of the renal osmolyte betaine. We show that the signals missed by transcriptome-wide MR are found, thanks to the increase in power conferred by integrating multiple omics layer. Simulation analyses show that with larger molecular QTL studies and in case of mediated effects, our multi-omics MR framework outperforms classical MR approaches designed to detect causal relationships between single molecular traits and complex phenotypes.

Organic osmolyte betaine mitigates the deleterious effects of Diclofenac in vivo in wistar albino rats

Organic osmolyte betaine mitigates the deleterious effects of Diclofenac in vivo in wistar albino rats

Skin-to-blood pH shift triggers metabolome and proteome global remodelling in Staphylococcus epidermidis.

Staphylococcus epidermidis is one of the most common bacteria of the human skin microbiota. Despite its role as a commensal, S. epidermidis has emerged as an opportunistic pathogen, associated with 80% of medical devices related infections. Moreover, these bacteria are extremely difficult to treat due to their ability to form biofilms and accumulate resistance to almost all classes of antimicrobials. Thus new preventive and therapeutic strategies are urgently needed. However, the molecular mechanisms associated with S. epidermidis colonisation and disease are still poorly understood. A deeper understanding of the metabolic and cellular processes associated with response to environmental factors characteristic of SE ecological niches in health and disease might provide new clues on colonisation and disease processes. Here we studied the impact of pH conditions, mimicking the skin pH (5.5) and blood pH (7.4), in a S. epidermidis commensal strain by means of next-generation proteomics and 1H NMR-based metabolomics. Moreover, we evaluated the metabolic changes occurring during a sudden pH change, simulating the skin barrier break produced by a catheter. We found that exposure of S. epidermidis to skin pH induced oxidative phosphorylation and biosynthesis of peptidoglycan, lipoteichoic acids and betaine. In contrast, at blood pH, the bacterial assimilation of monosaccharides and its oxidation by glycolysis and fermentation was promoted. Additionally, several proteins related to virulence and immune evasion, namely extracellular proteases and membrane iron transporters were more abundant at blood pH. In the situation of an abrupt skin-to-blood pH shift we observed the decrease in the osmolyte betaine and changes in the levels of several metabolites and proteins involved in cellular redoxl homeostasis. Our results suggest that at the skin pH S. epidermidis cells are metabolically more active and adhesion is promoted, while at blood pH, metabolism is tuned down and cells have a more virulent profile. pH increase during commensal-to-pathogen conversion appears to be a critical environmental signal to the remodelling of the S. epidermidis metabolism toward a more pathogenic state. Targeting S. epidermidis proteins induced by pH 7.4 and promoting the acidification of the medical device surface or surrounding environment might be new strategies to treat and prevent S. epidermidis infections.

Osmoregulatory Role of Betaine and Betaine/γ-Aminobutyric Acid Transporter 1 in Post-Traumatic Syringomyelia.

Syringomyelia (SM) is primarily characterized by the formation of a fluid-filled cyst that forms in the parenchyma of the spinal cord following injury or other pathology. Recent omics studies in animal models have identified dysregulation of solute carriers, channels, transporters, and small molecules associated with osmolyte regulation during syrinx formation/expansion in the spinal cord. However, their connections to syringomyelia etiology are poorly understood. In this study, the biological functions of the potent osmolyte betaine and its associated solute carrier betaine/γ-aminobutyric acid (GABA) transporter 1 (BGT1) were studied in SM. First, a rat post-traumatic SM model was used to demonstrate that the BGT1 was primarily expressed in astrocytes in the vicinity of syrinxes. In an in vitro system, we found that astrocytes uptake betaine through BGT1 to regulate cell size under hypertonic conditions. Treatment with BGT1 inhibitors, especially NNC 05-2090, demonstrated midhigh micromolar range potency in vitro that reversed the osmoprotective effects of betaine. Finally, the specificity of these BGT1 inhibitors in the CNS was demonstrated in vivo, suggesting feasibility for targeting betaine transport in SM. In summary, these data provide an enhanced understanding of the role of betaine and its associated solute carrier BGT1 in cell osmoregulation and implicates the active role of betaine and BGT1 in syringomyelia progression.

Comparative genomic analysis of obligately piezophilic Moritella yayanosii DB21MT-5 reveals bacterial adaptation to the Challenger Deep, Mariana Trench.

Hadal trenches are the deepest but underexplored ecosystems on the Earth. Inhabiting the trench bottom is a group of micro-organisms termed obligate piezophiles that grow exclusively under high hydrostatic pressures (HHP). To reveal the genetic and physiological characteristics of their peculiar lifestyles and microbial adaptation to extreme high pressures, we sequenced the complete genome of the obligately piezophilic bacterium Moritella yayanosii DB21MT-5 isolated from the deepest oceanic sediment at the Challenger Deep, Mariana Trench. Through comparative analysis against pressure sensitive and deep-sea piezophilic Moritella strains, we identified over a hundred genes that present exclusively in hadal strain DB21MT-5. The hadal strain encodes fewer signal transduction proteins and secreted polysaccharases, but has more abundant metal ion transporters and the potential to utilize plant-derived saccharides. Instead of producing osmolyte betaine from choline as other Moritella strains, strain DB21MT-5 ferments on choline within a dedicated bacterial microcompartment organelle. Furthermore, the defence systems possessed by DB21MT-5 are distinct from other Moritella strains but resemble those in obligate piezophiles obtained from the same geographical setting. Collectively, the intensive comparative genomic analysis of an obligately piezophilic strain Moritella yayanosii DB21MT-5 demonstrates a depth-dependent distribution of energy metabolic pathways, compartmentalization of important metabolism and use of distinct defence systems, which likely contribute to microbial adaptation to the bottom of hadal trench.

Effect of osmolytes on in-vitro aggregation properties of peptides derived from TGFBIp

Protein aggregation has been one of the leading triggers of various disease conditions, such as Alzheimer’s, Parkinson’s and other amyloidosis. TGFBI-associated corneal dystrophies are protein aggregation disorders in which the mutant TGFBIp aggregates and accumulates in the cornea, leading to a reduction in visual acuity and blindness in severe cases. Currently, the only therapy available is invasive and there is a known recurrence after surgery. In this study, we tested the inhibitory and amyloid dissociation properties of four osmolytes in an in-vitroTGFBI peptide aggregation model. The 23-amino acid long peptide (TGFBIp 611–633 with the mutation c.623 G>R) from the 4th FAS-1 domain of TGFBIp that rapidly forms amyloid fibrils was used in the study. Several biophysical methods like Thioflavin T (ThT) fluorescence, Circular Dichroism (CD), fluorescence microscopy and Transmission electron microscopy (TEM) were used to study the inhibitory and amyloid disaggregation properties of the four osmolytes (Betaine, Raffinose, Sarcosine, and Taurine). The osmolytes were effective in both inhibiting and disaggregating the amyloid fibrils derived from TGFBIp 611–633 c.623 G>R peptide. The osmolytes did not have an adverse toxic effect on cultured human corneal fibroblast cells and could potentially be a useful therapeutic strategy for patients with TGFBIp corneal dystrophies.

Betaine/GABA transporter-1 (BGT-1) deficiency in mouse prevents acute liver failure in vivo and hepatocytes apoptosis in vitro

Betaine/γ-aminobutyric acid (GABA) transporter 1 (BGT-1 or Slc6a12) is a transporter for the neurotransmitter GABA and osmolyte betaine. To date, most studies on BGT-1 have focused on its functions in the nervous system and renal osmotic homeostasis. Despite its dominant distribution in the liver, the function of BGT-1 in hepatic physiology or disease remains unknown. Here, we report that BGT-1 was significantly downregulated in patients with liver failure as well as in mice with experimental acute liver failure (ALF). Furthermore, mice deficient in BGT-1 showed significant resistance to ALF compared with wild type (WT) mice, manifesting as improved survival rate, reduced alanine transaminase/aspartate aminotransferase levels, better histopathological symptoms and fewer apoptotic cells in the liver. Similarly, in primary hepatocytes, BGT-1 deficiency or treatment with a BGT-1 inhibitor, NNC 05-2090, attenuated TNF-α mediated apoptosis. In addition, BGT-1 deficiency or dosing with NNC 05-2090 stimulated the expression of the anti-apoptotic gene, c-Met in the liver, suggesting the involvement of c-Met in the function on hepatocytes of BGT-1 apoptosis. Our findings suggest BGT-1 is a promising candidate drug target to prevent and treat hepatocyte apoptosis related diseases, such as ALF.

Betaine and Antioxidants Improve Growth Performance, Breast Muscle Development and Ameliorate Thermoregulatory Responses to Cyclic Heat Exposure in Broiler Chickens.

Simple SummaryThe physiological and metabolic responses of broiler chickens to control thermoregulation during heat stress divert energy from efficient production in addition to increasing morbidity and mortality. Therefore, heat stress amelioration strategies may improve the productivity of poultry meat production over the summer months and in tropical regions. Increasingly, low-cost feed additives are being investigated as potential amelioration strategies against heat stress. Scholars have investigated the effects of betaine alone on growth performance and gut physiology, while a small number of studies have been made regarding the impacts of the combination of betaine and antioxidants. Therefore, this study was conducted to investigate the effects of the osmolyte betaine and selenium and vitamin E supplementation on growth performance, physiological responses, and gut physiology in broiler chickens exposed to cyclic heat stress.Heat stress (HS) is an environmental stressor challenging poultry production and requires a strategy to cope with it. A total of 288-day-old male broiler chicks were fed with one of the following diets: basal diet, basal with betaine (BET), or with selenium and vitamin E (AOX), or with a combination of BET and AOX, under thermoneutral and cyclic HS. Results showed that HS reduced average daily feed intake (ADFI) (p = 0.01) and average daily gain (ADG) (p < 0.001), and impaired feed conversion ratio (FCR) (p = 0.03) during rearing period (0–42 day). BET increased ADG (p = 0.001) and decreased FCR (p = 0.02), whereas AOX had no effects. Breast muscle weight was decreased by HS (p < 0.001) and increased by BET (p < 0.001). Rectal temperature was increased by HS (p < 0.001) and improved by BET overall. Respiration rate was increased by HS (p < 0.001), but BET decreased it during HS (p = 0.04). Jejunum transepithelial resistance was reduced by HS and had no effect on permeability whereas BET increased jejunum permeability (p = 0.013). Overall, the reductions in ADG of broiler chickens during HS were ameliorated by supplementation with BET, with much of the increase in ADG being breast muscle.

Osmolyte taurine induction in UVA exposed human retinal pigment epithelial cells

Aim: To explore the osmolytes expression in ultraviolet (UVA) stressed human retinal pigment epithelial cells.Methods: Osmolyte transporters and vascular endothelial growth factor (VEGF) messenger RNA (mRNA) were determined by real time polymerase chain reaction (PCR). Osmolyte uptake was measured by radioimmunoassay. VEGF concentrations were determined by immunoassay and enzyme-linked immunosorbent assay (ELISA). Osmolyte taurine transporter (TAUT) were silenced by siRNA technology.Results: Hypertonicity accelerated osmolyte betaine uptake, myoinositol uptake, and taurine uptake, compared to normotonic stress. UVA irradiation also accelerated osmolyte transporters expression and osmolytes uptake. Especially, osmolyte taurine remarkably inhibited VEGF release induced by UVA irradiation. VEGF in the UVA stressed retinal pigment epithelial cell supernatant was accumulated slow after taurine preincubation. VEGF expression increased significantly in UVA-stressed cells after TAUT silencing. Moreover, taurine reduced the VEGF level in human ocular aqueous humor.Conclusion: The inhibition of VEGF by osmolyte taurine plays the crucial role in retina adaption to UVA irradiation.

Small Angle Neutron Scattering Studies of R67 Dihydrofolate Reductase, a Tetrameric Protein with Intrinsically Disordered N-Termini.

R67 dihydrofolate reductase (DHFR) is a homotetramer with a single active site pore and no sequence or structural homology with chromosomal DHFRs. The R67 enzyme provides resistance to trimethoprim, an active site-directed inhibitor of Escherichia coli DHFR. Sixteen to twenty N-terminal amino acids are intrinsically disordered in the R67 dimer crystal structure. Chymotrypsin cleavage of 16 N-terminal residues results in an active enzyme with a decreased stability. The space sampled by the disordered N-termini of R67 DHFR was investigated using small angle neutron scattering. From a combined analysis using molecular dynamics and the program SASSIE (http://www.smallangles.net/sassie/SASSIE_HOME.html), the apoenzyme displays a radius of gyration (Rg) of 21.46 ± 0.50 Å. Addition of glycine betaine, an osmolyte, does not result in folding of the termini as the Rg increases slightly to 22.78 ± 0.87 Å. SASSIE fits of the latter SANS data indicate that the disordered N-termini sample larger regions of space and remain disordered, suggesting they might function as entropic bristles. Pressure perturbation calorimetry also indicated that the volume of R67 DHFR increases upon addition of 10% betaine and decreased at 20% betaine because of the dehydration of the protein. Studies of the hydration of full-length R67 DHFR in the presence of the osmolytes betaine and dimethyl sulfoxide find around 1250 water molecules hydrating the protein. Similar studies with truncated R67 DHFR yield around 400 water molecules hydrating the protein in the presence of betaine. The difference of ∼900 waters indicates the N-termini are well-hydrated.

Cap-independent protein synthesis is enhanced by betaine under hypertonic conditions

Protein synthesis is one of the main cellular functions inhibited during hypertonic challenge. The subsequent accumulation of the compatible osmolyte betaine during the later adaptive response allows not only recovery of translation but also its stimulation. In this paper, we show that betaine modulates translation by enhancing the formation of cap-independent 48 S pre-initiation complexes, leaving cap-dependent 48 S pre-initiation complexes basically unchanged. In the presence of betaine, CrPV IRES- and sodium-dependent neutral amino acid transporter-2 (SNAT2) 5′-UTR-driven translation is 2- and 1.5-fold stimulated in MCF7 cells, respectively. Thus, betaine could provide an advantage in translation of messengers coding for proteins implicated in the response of cells to different stressors, which are often recognized by ribosomal 40 S subunit through simplified cap-independent mechanisms.

Effects of dietary supplementation of the osmolyte betaine on growing pig performance and serological and hematological indices during thermoneutral and heat-stressed conditions.

The present study was designed to evaluate the effects of dietary betaine on pig performance and serological and hematological indices during thermoneutral and heat-stressed conditions. Individually housed pigs ( = 64; 39.0 ± 1.5 kg BW) were assigned within weight blocks and sex to 1 of 8 treatments. Treatments consisted of 2 environmental conditions (thermoneutral or heat-stressed) and 4 levels of betaine (0, 0.10, 0.15, and 0.20%). Room temperatures followed a daily pattern with a low of 14°C and a high of 21°C for the thermoneutral environment and a low of 28°C and a high of 35°C for the heat-stressed environment. Experimental diets were fed from d -7 (7 d prior to imposing temperature treatments; constant 21°C) until 28. Respiration rate and rectal temperature were measured on d 0, 1, 2, 3, 7, 14, 21, and 28, and blood samples were collected on d 3 and 28. Heat stress reduced ( ≤ 0.008) ADG (0.710 vs. 0.822 kg/d) and ADFI (1.81 vs. 2.27 kg/d) and increased G:F ( = 0.036; 0.391 vs. 0.365). Betaine tended to quadratically increase G:F ( = 0.071; 0.377, 0.391, 0.379, and 0.366 for 0, 0.10, 0.15, and 0.20% betaine, respectively), regardless of environment. Heat stress increased ( ≤ 0.001) respiration rate (48 vs. 23 breaths/30 s) and rectal temperature (39.47 vs. 38.94°C) throughout d 1 to 28. Betaine at 0.10% reduced rectal temperature in heat-stressed pigs but not in control pigs (interaction, = 0.040). Heat stress increased serum cysteine and triglycerides and reduced Ca, alkaline phosphatase, and lipase, regardless of day of sampling ( ≤ 0.048). Heat stress increased serum creatine phosphokinase (CPK) and K and reduced osmolarity, Na, urea N, methionine, homocysteine, the albumin:globulin ratio, and blood eosinophil count on d 3 but not on d 28 (interaction, ≤ 0.013). Heat stress increased serum Mg, globulin, creatinine, amylase, and γ-glutamyltranspeptidase and reduced , the urea N:creatinine ratio, alanine aminotransferase, NEFA, hemoglobin, hematocrit, and red blood cells on d 28 but not on d 3 (interaction, ≤ 0.034). Betaine increased serum osmolarity and NEFA and reduced CPK and K on d 3 but not on d 28 (interaction, ≤ 0.060) and increased serum creatinine and reduced amylase on d 28 but not on d 3 (interaction ≤ 0.057). Heat stress reduced growth, disturbed ion balance, and increased markers of muscle damage. Betaine had a minor impact on alleviating heat stress with the possible exception of early days of heat exposure. The beneficial effect of betaine was diminished by pig adaptation.

Comparison of Enzymatic Traits between Native and Recombinant Glycine Sarcosine N-Methyltransferase from Methanohalophilus portucalensis FDF1T.

The halophilic methanoarchaeon Methanohalophilus portucalensis FDF1T possesses the ability to synthesize the osmolyte betaine from its precursor, glycine, in response to extracellular salt stress through a three-step transmethylation process. Analysis of recombinant glycine sarcosine N-methyltransferase (rGSMT) and recombinant sarcosine dimethylglycine N-methyltransferase (rSDMT) from Escherichia coli indicated that betaine synthesis is rate-limited by rGSMT and is constitutively activated by rSDMT. Therefore, it is of interest to purify native GSMT from Methanohalophilus portucalensis to further compare its enzymatic characteristics and kinetics with rGSMT. In this study, native GSMT was purified through DEAE ion exchange and gel filtration chromatography with 95% purity. The enzymatic characteristics of GSMT and rGSMT showed similar trends of activities that were activated by high concentrations of monovalent cations. Both were feedback-inhibited by the end product, betaine, and competitively inhibited by S-adenosylhomocysteine (SAH). Native GSMT was 2-fold more sensitive to SAH than rGSMT. Notably, comparison of the kinetic parameters illustrated that the turnover rate of glycine methylation of GSMT was promoted by potassium ions, whereas rGSMT was activated by increasing protein-glycine binding affinity. These results suggest that GSMT and rGSMT may have different levels of post-translational modifications. Our preliminary mass spectrometry evidence indicated that there was no detectable phosphosite on GSMT after the complicated purification processes, whereas purified rGSMT still possessed 23.1% of its initial phosphorylation level. We believe that a phosphorylation-mediated modification may be involved in the regulation of this energy consuming betaine synthesis pathway during the stress response in halophilic methanoarchaea.

Dietary Betaine Impacts the Physiological Responses to Moderate Heat Conditions in a Dose Dependent Manner in Sheep.

Simple SummaryHeat stress in sheep initiates physiological methods to dissipate heat that result in decreased production. This study investigated the use of a dietary supplement, the osmolyte betaine fed at two doses (2 or 4 g/day), on the physiological responses to heat in sheep. Heat exposure initiated physiological responses such as an increased rectal temperature and respiration rate as expected, while betaine supplemented at 2 g/day ameliorated these responses. Thus, dietary betaine supplementation may have beneficial effects for sheep exposed to heat.Heat exposure (HE) results in decreased production in ruminant species and betaine is proposed as a dietary mitigation method. Merino ewes (n = 36, 40 kg, n = 6 per group) were maintained at thermoneutral (TN, n = 18, 21 °C) or cyclical HE (n = 18, 18–43 °C) conditions for 21 days, and supplemented with either 0 (control), 2 or 4 g betaine/day. Sheep had ad libitum access to water and were pair fed such that intake of sheep on the TN treatment matched that of HE animals. Heart rate (HR), respiration rate (RR), rectal (TR) and skin temperatures (TS) were measured 3 times daily (0900 h, 1300 h, 1700 h). Plasma samples were obtained on 8 days for glucose and NEFA analysis. The HE treatment increased TR by 0.7 °C (40.1 vs. 39.4 °C for HE and TN respectively p < 0.001), TS by +1.8 °C (39.3 vs. 37.5 °C, p < 0.001) and RR by +46 breaths/min (133 vs. 87 breaths/min, p < 0.001) compared to TN. The 2 g betaine/day treatment decreased TR (39.8, 39.6 and 39.8 °C, p < 0.001), TS (38.7, 38.0 and 38.5 °C, p < 0.001) and RR (114, 102 and 116 breaths/min for control, 2 and 4 g betaine/day, p < 0.001) compared to control. Betaine supplementation decreased plasma NEFA concentrations by ~25 μM (80, 55 and 54 μmol/L for 0, 2 and 4 g/day respectively, p = 0.05). These data indicate that dietary betaine supplementation at 2 g betaine/day provides improvements in physiological responses typical of ewes exposed to heat stress and may be a beneficial supplement for the management of sheep during summer.

Population-specific renal proteomes of marine and freshwater three-spined sticklebacks

Quantitative proteomics was used to reveal biochemical differences in kidneys of marine and freshwater three-spined sticklebacks. More than 1500 unambiguous proteins were identified, 106 of which are robustly co-translationally modified. Amino-terminal acetylation sites for 94 and proline hydroxylation sites for 12 proteins, including 4 protein disulfide isomerases having the consensus motif APWCGHCK, were determined. More than 1500 proteins were quantified by LC–MS/MS yielding 120 proteins with consistent population-specific abundance differences. Twenty-five of these were selected for validation by data-independent acquisition (DIA) and spectral library based MS2 quantitation. A dense biochemical network was revealed, which promotes the synthesis of the organic osmolytes betaine, sorbitol, trimethylamine oxid (TMAO), and urea. It contains 33 of 49 proteins that are elevated in marine compared to freshwater sticklebacks, including the most highly elevated proteins (dimethylaniline monooxygenase, alanine-glyoxylate aminotransferase, glycine N-methyltransferase). Freshwater stickleback kidneys contain elevated levels of proteolytic, cytoskeletal, extracellular matrix, and calcium signaling proteins. Proteins that are most elevated in freshwater sticklebacks are ES1 protein homolog, apoptosis-associated speck-like protein containing a CARD and caspase 1. Protein-abundance network analysis demonstrates significantly higher levels of synchronized abundance control in marine sticklebacks. The significance of these findings for biochemical diversification of renal function in marine and FW sticklebacks is discussed.

The organic osmolyte betaine induces keratin 2 expression in rat epidermal keratinocytes — A genome-wide study in UVB irradiated organotypic 3D cultures

The moisturizing and potentially protective properties of the organic osmolyte betaine (trimethylglycine) have made it an attractive component for skin care products. Its wide use despite the lack of comprehensive studies addressing its specific effects in skin led us to characterize the molecular targets of betaine in keratinocytes and to explore, whether it modifies the effects of acute UVB exposure.Genome-wide expression analysis was performed on organotypic cultures of rat epidermal keratinocytes, treated either with betaine (10mM), UVB (30mJ/cm2) or their combination. Results were verified with qRT-PCR, western blotting and immunohistochemistry. Additionally, cell proliferation and differentiation were analyzed.Among the 89 genes influenced by betaine, the differentiation marker keratin 2 showed the highest upregulation, which was also confirmed at protein level. Expression of Egr1, a transcription factor, and Purkinje cell protein 4, a regulator of Ca2+/calmodulin metabolism, also increased, while downregulated genes included several ion-channel components, such as Fxyd2.Bioinformatics analyses suggest that genes modulated by betaine are involved in DNA replication, might counteract UV-induced processes, and include many targets of transcription factors associated with cell proliferation and differentiation.Our results indicate that betaine controls unique gene expression pathways in keratinocytes, including some involved in differentiation.

Synergistic effect of antioxidant system and osmolyte in hydrogen sulfide and salicylic acid crosstalk-induced heat tolerance in maize (Zea mays L.) seedlings

Salicylic acid (SA), is a plant hormone with multifunction that is involved in plant growth, development and the acquisition of stress tolerance. Hydrogen sulfide (H2S) is emerging similar functions, but crosstalk between SA and H2S in the acquisition of heat tolerance is not clear. Our recent study firstly reported that SA treatment enhanced the activity of L-cysteine desulfhydrase (L-DES), a key enzyme in H2S biosynthesis, followed by induced endogenous H2S accumulation, which in turn improved the heat tolerance of maize seedlings.1 In addition, NaHS, a H2S donor, enhanced SA-induced heat tolerance, while its biosynthesis inhibitor DL-propargylglycine (PAG) and scavenger hydroxylamine (HT) weakened SA-induced heat tolerance. Also, NaHS had no significant effect on SA accumulation and its biosynthesis enzymes phenylalanine ammonia lyase (PAL) and benzoic-acid-2-hydroxylase (BA2H) activities, as well as significant difference was not observed in NaHS-induced heat tolerance of maize seedlings by SA biosynthesis inhibitors paclobutrazol (PAC) and 2-aminoindan-2-phosph- onic acid (AIP) treatment.1 Further study displayed that SA induced osmolytes (proline, betaine and trehalose) accumulation and enhancement in activity of antioxidant system in maize seedlings. These results showed that antioxidant system and osmolyte play a synergistic role in SA and H2S crosstalk-induced heat tolerance of maize seedlings.

Seed germination, plant growth and physiological responses of Salsola ikonnikovii to short-term NaCl stress

Salsola ikonnikovii (Chenopodiaceae), a drought-tolerant plant species that is distributed in sand or light-saline soil in Xinjiang, China, produces seeds (fruits) with attached winged perianths. To study the role of the wing in seed germination under salt stress and to further investigate the growth and physiological responses of the plants to salt stress, the germination behaviour of S. ikonnikovii was determined after winged and non-winged seeds were treated with 0–1000 mmol · L−1 NaCl. Several parameters of two-month old plants that had been treated with NaCl for three weeks were measured. The results revealed that the winged perianths limited germination but protected the seeds from salt damage. The growth of the plants was stimulated by lower concentrations of salt (≤100 mmol · L−1 NaCl), while increasing salt concentrations inhibited growth. The level of reactive oxygen species and malondialdehyde increased significantly at high concentrations of salt. Correspondingly, concentrations of the osmolytes proline, betaine, and soluble sugars, and the activities of antioxidative enzymes (catalase, peroxidase and superoxide dismutase) increased, but the levels of non-enzymatic antioxidants (carotenoids, glutathione) were significantly reduced at high salt concentrations. These results imply that osmotic adjustment and the antioxidative system may work synergistically to ensure that a plant grows normally under high salt concentrations.

Characterization and Regulation of the Osmolyte Betaine Synthesizing Enzymes GSMT and SDMT from Halophilic Methanogen Methanohalophilus portucalensis

The halophilic methanoarchaeon Methanohalophilus portucalensis can synthesize the osmolyte betaine de novo in response to extracellular salt stress. Betaine is generated by the stepwise methylation of glycine to form sarcosine, N, N-dimethylglycine and betaine by using S-adenosyl-L-methionine (AdoMet) as the methyl donor. The complete gene cluster of Mpgsmt-sdmt was cloned from Southern hybridization and heterologous expressed in E. coli respectively. The recombinant MpGSMT and MpSDMT both retained their in vivo functional activities in E. coli BL21(DE3)RIL to synthesize and accumulate betaine and conferred elevated survival ability in betaine transport deficient mutant E. coli MKH13 under high salt stress. The dramatic activating effects of sodium and potassium ions on the in vitro methyltransferase activities of MpGSMT, but not MpSDMT or bacterial GSMT and SDMT, revealed that GSMT from halophilic methanoarchaeon possesses novel regulate mechanism in betaine biosynthesis pathway. The circular dichroism spectra showed the fluctuated peaks at 206 nm were detected in the MpGSMT under various concentrations of potassium or sodium ions. This fluctuated difference may cause by a change in the β-turn structure located at the conserved glycine- and sarcosine-binding residue Arg167 of MpGSMT. The analytical ultracentrifugation analysis indicated that the monomer MpGSMT switched to dimeric form increased from 7.6% to 70% with KCl concentration increased from 0 to 2.0 M. The level of potassium and sodium ions may modulate the substrate binding activity of MpGSMT through the conformational change. Additionally, MpGSMT showed a strong end product, betaine, inhibitory effect and was more sensitive to the inhibitor AdoHcy. The above results indicated that the first enzymatic step involved in synthesizing the osmolyte betaine in halophilic archaea, namely, GSMT, may also play a major role in coupling the salt-in and compatible solute (osmolyte) osmoadaptative strategies in halophilic methanogens for adapting to high salt environments.