trans-4-hydroxy-β-prolinebetaine,a new betaine fromFurcellaria lumbricalis

From the red marine alga Furcellaria lumbricalis (Huds.) Lamour, a novel betaine has been isolated and characterised from infra-red and nuclear magnetic resonance spectroscopic and mass spectrometric data astrans-4-hydroxy-β-prolinebetaine.     

Extrinsic protein stabilization by the naturally occurring osmolytes β-hydroxyectoine and betaine

Abstract Thermodynamic aspects of protein stabilization by two widespread naturally occurring osmolytes, β-hydroxyectoine and betaine, were studied using differential scanning calorimetry (DSC) and bovine ribonuclease A (RNase A) as a model protein. The osmolyte β-hydroxyectoine purified from Marinococcus was found to be a very efficient stabilizer. At a concentration of 3 M it increased the melting temperature of RNase A (T _m ) by more than 12 K and gave rise to a stability increase of 10.6 kJ/mol at room temperature. The heat capacity difference between the folded and unfolded state (ΔC _p ) was found to be significantly increased. Betaine stabilized RNase A only at concentrations less than 3 M. Also, here ΔC _p was found to be increased. Calculation of the number of water molecules that additionally bind to unfolded RNase A resulted in surprisingly low numbers for both osmolytes. The significant stabilization of RNase A by β-hydroxyectoine makes this osmolyte an interesting stabilizer in biotechnological processes in which enzymes are applied in the presence of denaturants or at high temperature. Received: November 16, 1998 / Accepted: March 18, 1999     

Is glycine betaine a non-compatible solute in higher plants that do not accumulate it?

Rape (Brassica napus L. var. oleifera cv. Samourai) leaf discs treated in vitro in the presence of glycine betaine (GB) exhibited very high accumulation of GB, suggesting the operation of a specific uptake system. When further subjected to osmotic upshocks by transfer to PEG 6000 media, the typical osmo-induced proline response of the discs was strongly inhibited. The level of this inhibition was quantitatively related to the amount of GB loaded in the tissues. In contrast, the soluble sugar content increased in GB-treated discs. Surprisingly, viability tests (i.e. membrane stability and 2,3,5-triphenyltetrazolium chloride reduction) indicated a destabilizing effect of GB in these tissues. This is at variance with the relative compatibility of sucrose and proline. In addition, the protein content was lower in GB-treated discs. This could be related to an inhibitory effect on protein synthesis, as demonstrated by radiolabelling of polypeptides with [³⁵S] amino acids. This effect was particularly pronounced on Rubisco large sub-unit synthesis and was still apparent under non-stress conditions. The GB treatment was also followed by the induction or up-regulation of a set of polypeptides, not seen under stress conditions, while the synthesis of osmo-induced polypeptides was not affected by GB. These novel effects of GB lead us to discuss the reasons for its incompatibility in leaf tissues of a non-GB-accumulating species.     

Exogenously supplied glycine betaine in spinach and rapeseed leaf discs: compatibility or non-compatibility?

CMS, cell membrane stability
GB, glycine betaine
PEG, polyethylene glycol
TTC, 2,3,5-triphenyltetrazolium chloride

When leaf discs of spinach ( Spinacia oleracea cv. Junius) and rapeseed ( Brassica napus var. oleifera cv. Samourai) were incubated in the light in the presence of glycine betaine (GB), they accumulated GB at a very high level. In comparison with the spinach leaf explants, the uptake of GB by rapeseed tissues was restricted, probably by the destabilizing effects exerted by GB in this plant material. In contrast, the viability of spinach leaf discs, as assessed by their capacity to reduce 2,3,5-triphenyltetrazolium chloride (TTC), was not affected, suggesting that the GB taken up was compatible in the leaf tissues of the GB accumulator. In rapeseed leaf discs treated with GB, chlorophyll loss as well as significant changes in polyamine content were induced, leading to a dramatic increase of the putrescine/(spermidine + spermine) ratio. In contrast, this ratio remained constant in the GB treated spinach explants, suggesting that spinach has the capacity to stabilize polyamine metabolism in the presence of high amounts of GB. The treatment of spinach leaf discs with GB prior to application of osmotic or salt shocks provided protection from stress. A weak capacity to accumulate proline under stress conditions was partially suppressed. The protein content decreased while the free amino acid level increased independently of the presence of GB. It is concluded that GB behaves as a true compatible solute in spinach, which is a typical GB accumulator, and that GB is damaging when loaded into the leaf tissues of rapeseed, which do not normally accumulate GB.     

The role of β-dimethylsulphoniopropionate,glycine betaine and homarine in the osmoacclimation of Platymonas subcordiformis

The tertiary sulphonium compound, -dimethylsulphoniopropionate (DMSP) and the quaternary ammonium compounds glycine betaine and homarine are important osmotica in Platymonas subcordiformis cells. Following hypersaline stresses the compounds were accumulated after a lag period of 3 h and equilibrium concentrations were reached 6 h later. In contrast to these organic solutes, mannitol was synthesised immediately and equilibrium concentrations were reached within 90 min. Hyposaline stresses induced losses of the organic solutes from the cells. The ions K⁺, Na⁺, Cl^- and the above organic solutes can account for the osmotic balance of the cells.Abbreviations DMSP -dimethylsulphoniopropionate - _i intracellular osmolality - _o extracellular osmolality     

Effects of hyperhomocysteinemia and betaine–homocysteine S-methyltransferase inhibition on hepatocyte metabolites and the proteome

Both cardiovascular disease and liver injury are major public health issues. Hyperhomocysteinemia has been linked to cardiovascular diseases, and defects in methyl group metabolism, often resulting in hyperhomocysteinemia, are among the key molecular events postulated to play a role in liver injury. We employed proteomics and metabolomics analyses of human hepatocytes in primary cell culture to explore the spectrum of proteins and associated metabolites affected by the disruption of methyl group metabolism. We treated the hepatocytes with homocysteine (Hcy, 0.1 mM and 2 mM) to follow the impact of hyperhomocysteinemia, and in parallel, we used a specific inhibitor of betaine–homocysteine S-methyltransferase (BHMT) to extend our understanding of the physiological functions of the enzyme. The major effect of BHMT inhibition was a 50% decrease in S-adenosylmethionine levels. The treatments with Hcy resulted in multiple changes in the metabolite levels depending on the treatment modality. The BHMT inhibition and 0.1 mM Hcy treatment induced only moderate changes in the hepatocyte proteome and secretome, while the changes induced by the 2 mM Hcy treatment were extensive. Phosphatidylethanolamine carboxykinase and ornithine aminotransferase were up-regulated about two fold indicating an intervention into metabolism. Cellular proliferation was suspended, secretome composition was changed and signs of apoptosis were discernible. We have detected fibrinogen gamma dimers, which might have a role as a potentially new biomarker of early liver injury. Finally, we have demonstrated the failed maturation of apolipoprotein A1, which might be a new mechanism of disruption of cholesterol efflux from tissues.     

The salt-induced ABC transporter Ota of the methanogenic archaeon Methanosarcina mazei Gö1 is a glycine betaine transporter

The genes encoding the three subunits of the primary ABC transporter Ota of the methanogenic archaeon Methanosarcina mazei G?1 were cloned in an expression vector (pBAD24) and transformed into the glycine betaine transport-negative mutant Escherichia coli MKH13. Ota was produced as demonstrated by Western blotting. Uptake studies revealed that Ota catalyzed the transport of glycine betaine in E. coli MKH13(pBAD-Ota) with a K(m) of 10+/-5 microM and a maximal velocity of 1.5+/-0.5 nmol min(-1) mg protein(-1). Transport was ATP dependent. Ota was activated by salinity gradients, but only marginally by sugar gradients across the membrane. Glycine betaine transport was inhibited to a small extent by an excess of dimethylglycin or proline betaine, but not by sarcosine or glycine.     

Intracellular salt and solute concentrations in Ectothiorhodospira marismortui: glycine betaine and Nα-carbamoyl glutamineamide as osmotic solutes

Ectothiorhodospira marismortui, a moderately halophilic purple sulfur bacterium from a hypersaline sulfur spring, contains glycine betaine and N-carbamoyl glutamineamide (CGA) as the main intracellular osmotic solutes, with sucrose as a minor component. The concentration of glycine betaine was found to increase with increasing salt concentration of the medium, from 0.47 M to 1.29 M in cells grown from 0.85 to 2.56 M NaCl, while the estimated CGA concentration rose from about 0.2 M to 0.5 M. The concentration of sucrose remained constant at a value of around 0.05 M. Intracellular sodium and potassium concentrations were relatively low (around 0.5 and 0.3 M, respectively, at an external NaCl concentration of 1.8 M). The concentration of the novel compound N-carbamoyl glutamineamide was enhanced when l-glutamine was added to the growth medium, suggesting that glutamine served as a precursor for the synthesis of the compound.Abbreviations CGA N-carbamoyl glutamineamide     

Determination of betaine metabolites and dimethylsulfoniopropionate in coral tissues using liquid chromatography–time-of-flight mass spectrometry and stable isotope-labeled internal standards

A convenient procedure for determination of seven betaine analogs and dimethylsulfoniopropionate (DMSP) in extracts of coral tissues using LC–MS stable isotope dilution is described. Extraction procedures were optimized for selective extraction of polar metabolites from coral tissues. The LC–MS protocol employed a pentafluorophenylpropyl (PFPP) column for HPLC separation, with chromatographic resolution of isobaric and isomeric zwitterionic metabolites optimized by adjusting the acidity of the mobile phase. A ternary gradient was used to exploit the unusual retention characteristics of cationic metabolites on the PFPP column, with incorporation of ammonium acetate in a later gradient stage promoting elution of more hydrophobic betaines which are retained at high organic content in the absence of ammonium acetate. We demonstrate that the new LC–MS based method provides accurate measurements from nanomolar to high micromolar concentrations, and can be applied for profiling of betaine metabolites and DMSP in corals or other aquatic organisms.     

Differential regulation of Ota and Otb, two primary glycine betaine transporters in the methanogenic archaeon methanosarcina mazei Gö1

Methanogenic archaea accumulate glycine betaine in response to hypersalinity, but the regulation of proteins involved, their mechanism of activation and regulation of the corresponding genes are largely unknown. Methanosarcina mazei differs from most other methanoarchaea in having two gene clusters both encoding a potential glycine betaine transporter, Ota and Otb. Western blot as well as quantitative real-time PCR revealed that Otb is not regulated by osmolarity. On the other hand, cellular levels of Ota increased with increasing salt concentrations. A maximum was reached at 300-500 mM NaCl. Ota concentrations reached a maximum 4 h after an osmotic upshock. Hyperosmolarity also caused an increase in cellular Ota concentrations. In addition to osmolarity Ota expression was regulated by the growth phase. Expression of Ota as well as transport of betaine was downregulated in the presence of glycine betaine.     

The effects of betaine treatment on rats fed an acute bolus of ethanol at 3 and 12 h post bolus: a microarray analysis

Betaine, a methyl donor active in methionine metabolism, is effective in preventing and reversing experimental alcohol liver disease. The metabolic and molecular biologic mechanisms involved in this prevention are only partially known. To further investigate how betaine modifies the effects of ethanol on the liver, rats were given an acute ethanol bolus with or without betaine and the results were compared to isocaloric dextrose-fed controls. Livers were subjected to microarray analysis, and functional pathways and individual gene expression changes were analyzed. Experimental groups were compared by Venn diagrams showing that both ethanol and betaine caused a change in the expression of a large number of genes indicating that the changes were global. The bio-informatic analysis showed that all the KEGG functional pathways were affected and mainly down regulated at 3 h post bolus when ethanol plus betaine were compared with ethanol-fed rats. The most profound effect of betaine was on the metabolic pathways both at 3 and 12 h post bolus. At 3 h, the changes in gene expression were mostly down regulated, but at 12 h, the changes were regulated equally up and down. This hypothesis-driven analysis showed that the effects of betaine on the effects of ethanol were partly transient.     

Evaluation of the roles of two compatible solutes,glycine betaine and trehalose,for the Acacia senegal–Sinorhizobium symbiosis exposed to drought stress

Plant and Soil - Acacia senegal (Mimosoideae) is a leguminous, nitrogen-fixing tree that grows in arid areas of Africa and the Near East. In this work, we studied the effects of drought stress on...     

Analysis of carnitine, betaine, γ-butyrobetaine, and separate short-chain acylcarnitines in pigeon plasma, crop milk and tissues by HPLC coupled with UV-detection

Carnitine, betaine, -butyrobetaine and separate carnitine esters are determined in blood plasma, crop milk, liver, heart and breast muscle of pigeons (Columba livia domestica) by means of HPLC with photometric detection at 245 nm. The method can be generally applied for samples of animal origin.     

Do dietary betaine and the antibiotic florfenicol influence the intestinal autochthonous bacterial community in hybrid tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis> ♀ × <Emphasis Type="Italic">O. aureus</Emphasis> ♂)?

The attractant betaine and the antibiotic growth promoter florfenicol are commonly used together in Chinese fresh water aquaculture, but there is no information about the effect of these two feed additive on the intestinal autochthonous bacterial community in hybrid tilapia (Oreochromis nilotica ♀ × O. aureas ♂). Hybrid tilapia (240 fish in total; 20 fish per net cage; three cages per group) were divided into four dietary groups: control group, no betaine or florfenical addition (CK); betaine group, 0.1% betaine added (B); florfenicol group, 0.002% florfenicol added (F); and combination group, 0.1% betaine and 0.002% florfenicol added together (BF). After 8 weeks of feeding, six fish from each cage were chosen randomly, the guts were sampled and pooled, and their intestinal autochthonous bacterial communities were analyzed by 16S rDNA-denaturing gradient gel electrophoresis. Enumeration of total gut autochthonous bacteria was analyzed by quantitative PCR with rpoB as the endogenous control. The results showed that the fish intestinal bacteria of group B were more diverse than that of CK, and that of F and BF groups was reduced in the total numbers and limited to certain bacterial species or genera (P < 0.05). This study revealed that betaine can promote some intestinal autochthonous bacteria, and florfenicol play a depressor role. When combined together, florfenicol may overshadow the effect of betaine on the predominant intestinal bacteria of tilapia.