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

Exposure to fluctuating salinity enhances free amino acid accumulation in Tigriopus californicus (Copepoda)

Intracellular concentrations of free amino acids (FAA) in the intertidal copepod Tigriopus californicus increase in response to hyperosmotic stress and decrease in response to hypo-osmotic stress. The purpose of this study was to determine if exposure to repeated bouts of osmotic stress resulted in changes in FAA accumulation or the degree of FAA retention in subsequent episodes. Five groups of T. californicus were exposed for 22 days to a fluctuating salinity regime which consisted of 24 h at 100% seawater followed by 24 h at either 90, 80, 70, 60 or 50% seawater (11 cycles). After the tenth exposure to 100% seawater, individuals from each treatment group were analyzed for alanine and proline concentration. Alanine and proline accumulation generally increased in proportion to the osmotic stress up to 60–100% seawater — additional osmotic stress failed to increase total accumulation. Prior exposure to fluctuating salinity increased the extent of alanine and proline retention observed upon transfer to a hypo-osmotic medium. The treatment group which had experienced the most extreme fluctuation (50–100% seawater) retained alanine and proline levels approximately 10- and 20-fold higher, respectively, than controls. A less severe salinity fluctuation was required to elicit this response for alanine (90–100% seawater) than for proline (60–100% seawater). Previous exposure to fluctuating salinity also resulted in increased alanine and proline accumulation in subsequent episodes of hyperosmotic stress. 24 h after transfer from 50 to 100% seawater, alanine and proline levels in the conditioned copepods were approximately 3- and 7-fold higher, respectively, than in copepods which had not been cycled. This facilitation in alanine and proline accumulation occurred after 10 and 11 cycles, respectively. Of the increased accumulation in alanine and proline, 7.0% and 22.5%, respectively, could be accounted for by the higher degree of FAA retention while under hypo-osmotic conditions.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47130/1/360_2004_Article_BF00692733.pd

The behaviour of domestic cats (Felis silvestris catus) in shelters and after rehoming

I dette studiet ble det utført en undersøkelse på to omplasseringssentre i Oslo, Norge, der atferden til 239 omplasserte katter ble registrert ved bruk av et spørreskjema. Skjemaet inkluderte 12 spørsmål om normalatferd, problematferd og anbefalinger angående plassering av katt i nytt hjem. Det andre spørreskjemaet ble sendt ut til eierne av de omplasserte kattene, rundt tre måneder etter adopsjon, 65 svarte. Spørreskjemaet til eier inkluderte 71 spørsmål og var tilsvarende det til omplasseringssentrene, men mer utdypende om miljøet, adopsjonen og forholdet til katten. Kattene var kastrerte, eller skulle bli det dersom de var for unge på tidspunktet ved adopsjon. Av eierne som svarte på undersøkelsen var 89 % kvinner, med en gjennomsnittlig alder på 40,8±9,9 år og 61,5 % av dem bodde på et tettsted. 70,8 % av kattene hadde mulighet til å gå helt fritt ute, mens 12,3 % kun var innekatter. De resterende hadde en begrenset utetilgang. Gjennomsnittet for antall katter i hvert hjem var på 1,42 og det tok rundt seks dager før katten slo seg til ro i det nye hjemmet sitt. 84,6 % hadde kattetoalett inne hele året. På omplasseringssentrene utførte kattene ‟positive” atferdstrekk ofte eller svært ofte, som å oppsøke fremmede. Atferdsproblemene ble stort sett utført aldri eller sjeldent, som aggressiv biting overfor mennesker. Det største atferdsproblemet var at katter klorte på inventar. I det nye hjemmet ble atferdstrekkende utført med høy hyppighet, med unntak av atferdene der katten oppsøker barn, ukjente katter, holder fremmede katter unna husstanden og jaktegenskaper. Av problematferd ble kloring på inventar utført oftest, fulgt av frykt eller angst overfor sterke lyder. Katter som hadde fri utetilgang utførte enkelte problematferder i større grad enn innekatter. Katter med fri utetilgang la f.eks. oftere fra seg avføring utenfor dokassen. I husstander med mer enn én katt, var det mindre utførte atferdsproblemer enn i husstander med kun én katt. Der det var mer enn én katt, viste kattene f.eks. mindre aggressiv biting og kloring overfor mennesker. Eiere med mer enn fem års erfaring med katt, hadde katter som sjeldnere viste problematferd. Ved en sammenligning mellom omplasseringskattene fra dette studiet og huskattene fra en ‟normal” bakgrunn i en tidligere undersøkelse utført av Westbye (1998), ble problematferd utført oftere av huskattene, for ni av elleve problematferder. De var f.eks. oftere aggressive overfor andre katter og urinmarkerte oftere inne. For begge gruppene var kloring på inventar den atferden som ble utført i størst grad. Da vi spurte eierne i hvor stor grad de ønsket at katten utførte ulike atferder, var det i halvparten av tilfellene en positiv korrelasjon med graden kattene faktisk utførte atferden, ellers ble atferden utført i en høyere grad enn ønsket. Etter rundt tre måneder med katten svarte 9,8 av 10 eiere at de var godt fornøyd eller svært godt fornøyd med katten. Med tanke på det følelsesmessige båndet mellom katt og eier, var 8 av 10 sterkt bundet eller svært sterkt bundet til katten sin. For det sosiale båndet mellom katt og eier lå gjennomsnittet på 8,43±1,48, på en skala fra 1 til 10. For valget av katt var personlighet av størst betydning, mens kattens utseende, bakgrunn og anbefaling fra senteret var av middels betydning. Dersom katten hovedsaklig ble valgt ut ifra utseende, var forholdet mellom eier og katt rangert dårligere. I det nye hjemmet ble atferdstrekkene utført i lavere grad enn på omplasseringssentrene. Generelt var det lite atferdsproblemer på omplasseringssentrene og i det nye hjemmet. Det var høyere forekomst av atferdsproblemer etter adopsjon. Atferden hadde forandret seg etter adopsjon, da det var noe mer atferdsproblemer og lavere hyppighet av normale atferder. Omplasseringskatter hadde mindre forekomst av atferdsproblemer enn huskatter som ikke hadde vært omplasserte. Kattene slo seg raskere til ro i det nye hjemmet, enn tidligere forsøk på omplasseringssenter. For de fleste atferdstrekkene opplevde eier at katten utførte dem i ønsket eller større grad. Tiden katten hadde vært hos eierne hadde ingen effekt på tilknytningsgraden mellom katt og eier. In this study a survey was conducted on two shelters in Oslo, Norway. The behaviour of 239 rehomed cats was registered on a questionnaire. The questionnaire included 12 questions regarding normal behaviour, problem behaviour and recommendations regarding the placement of a cat in the new home. The second questionnaire was sent out to the owners of the rehomed cats, about three months after the adoption, 65 responded. The questionnaire to the owner included 71 questions and was similar to the questions from the shelters, but more detailed about the environment, adoption and the relationship to the cat. The cats were neutered, or would it be if they were too young at the time of the adoption. 89 % of the respondents were female and the average age of the respondents were 40,8±9.9 years. 61,5 % of them lived in a highly populated area and 70,8 % of cats had the opportunity to walk freely outside, while only 12,3 % were kept as indoor cats. The remaining had a limited access to the outdoors. The average number of cats in each home was 1,42, and it took about six days before the cat settled down in their new home. 84,6 % had access to a litter box inside all year. On the shelters cats performed "positive" behavioral traits often or very often, like approaching strangers. The behavioural problems mostly occurred never or rarely, like aggressive biting towards humans. Scratching the inventory was the biggest behaviour problem. In the new home the behavioral traits were performed with high frequency, with the exception being when the cat approached children and unfamiliar cats, kept other cats away from the household and hunting. The most frequently executed problem behaviour was cats scratching the inventory, followed by fear and anxiety towards loud noises. Cats with free access to the outdoors showed problem behaviour to a greater extent than indoor cats. They would more often defecate outside the litter box. There were less behaviour problems in households with more than one cat, for example there were less aggressive biting and scratching towards humans. Owners, who had more than five years of experience with cats, observed less behaviour problems. When comparing the shelter cats from this study to cats from a previous study with a “normal” background, nine out of eleven behaviour problems were performed more often by the “normal” cats. They were more aggressive towards other cats and would more often urine mark inside. Scratching on the inventory was performed most by both of the groups.When asked about how much owners preferred certain behaviour traits in their cats, the response had a positive correlation with how often the cats executed the behaviour in half of the cases. About three months after the adoption 9,8 of 10 owners were satisfied or very satisfied with their cats. The owners scored their emotional bond to their cats strongly attached or very strongly attached in 8 out of 10 cases. The social bond between owner and cat had on a scale from 1 to 10, a mean of 8,43±1,48. When choosing a cat the owners put more weight on the cats’ personality, while appearance, background and recommendation from the shelter were of medium importance. Cats mainly chosen by their appearance, would have a less successful relationship with their owners. Behaviour traits were performed less in the cat’s new homes than at the shelters. Generally there were few behaviour problems at the shelters and in the new home. There was a higher frequency of behaviour problems after an adoption. The behaviour had changed after the adoption. The behaviour problems increased and the behaviour traits decreased. Cats from a shelter showed less of behaviour problems than cats with ‟normal” background. The cats settled faster in the new home, than former studies done at shelters. Time spent living together had no effect on the degree of attachment between cat and owner

Atferd hos katter (Felis silvestris catus) på omplasseringssentre og etter adopsjon

I dette studiet ble det utført en undersøkelse på to omplasseringssentre i Oslo, Norge, der atferden til 239 omplasserte katter ble registrert ved bruk av et spørreskjema. Skjemaet inkluderte 12 spørsmål om normalatferd, problematferd og anbefalinger angående plassering av katt i nytt hjem. Det andre spørreskjemaet ble sendt ut til eierne av de omplasserte kattene, rundt tre måneder etter adopsjon, 65 svarte. Spørreskjemaet til eier inkluderte 71 spørsmål og var tilsvarende det til omplasseringssentrene, men mer utdypende om miljøet, adopsjonen og forholdet til katten. Kattene var kastrerte, eller skulle bli det dersom de var for unge på tidspunktet ved adopsjon. Av eierne som svarte på undersøkelsen var 89 % kvinner, med en gjennomsnittlig alder på 40,8±9,9 år og 61,5 % av dem bodde på et tettsted. 70,8 % av kattene hadde mulighet til å gå helt fritt ute, mens 12,3 % kun var innekatter. De resterende hadde en begrenset utetilgang. Gjennomsnittet for antall katter i hvert hjem var på 1,42 og det tok rundt seks dager før katten slo seg til ro i det nye hjemmet sitt. 84,6 % hadde kattetoalett inne hele året. På omplasseringssentrene utførte kattene ‟positive” atferdstrekk ofte eller svært ofte, som å oppsøke fremmede. Atferdsproblemene ble stort sett utført aldri eller sjeldent, som aggressiv biting overfor mennesker. Det største atferdsproblemet var at katter klorte på inventar. I det nye hjemmet ble atferdstrekkende utført med høy hyppighet, med unntak av atferdene der katten oppsøker barn, ukjente katter, holder fremmede katter unna husstanden og jaktegenskaper. Av problematferd ble kloring på inventar utført oftest, fulgt av frykt eller angst overfor sterke lyder. Katter som hadde fri utetilgang utførte enkelte problematferder i større grad enn innekatter. Katter med fri utetilgang la f.eks. oftere fra seg avføring utenfor dokassen. I husstander med mer enn én katt, var det mindre utførte atferdsproblemer enn i husstander med kun én katt. Der det var mer enn én katt, viste kattene f.eks. mindre aggressiv biting og kloring overfor mennesker. Eiere med mer enn fem års erfaring med katt, hadde katter som sjeldnere viste problematferd. Ved en sammenligning mellom omplasseringskattene fra dette studiet og huskattene fra en ‟normal” bakgrunn i en tidligere undersøkelse utført av Westbye (1998), ble problematferd utført oftere av huskattene, for ni av elleve problematferder. De var f.eks. oftere aggressive overfor andre katter og urinmarkerte oftere inne. For begge gruppene var kloring på inventar den atferden som ble utført i størst grad. Da vi spurte eierne i hvor stor grad de ønsket at katten utførte ulike atferder, var det i halvparten av tilfellene en positiv korrelasjon med graden kattene faktisk utførte atferden, ellers ble atferden utført i en høyere grad enn ønsket. Etter rundt tre måneder med katten svarte 9,8 av 10 eiere at de var godt fornøyd eller svært godt fornøyd med katten. Med tanke på det følelsesmessige båndet mellom katt og eier, var 8 av 10 sterkt bundet eller svært sterkt bundet til katten sin. For det sosiale båndet mellom katt og eier lå gjennomsnittet på 8,43±1,48, på en skala fra 1 til 10. For valget av katt var personlighet av størst betydning, mens kattens utseende, bakgrunn og anbefaling fra senteret var av middels betydning. Dersom katten hovedsaklig ble valgt ut ifra utseende, var forholdet mellom eier og katt rangert dårligere. I det nye hjemmet ble atferdstrekkene utført i lavere grad enn på omplasseringssentrene. Generelt var det lite atferdsproblemer på omplasseringssentrene og i det nye hjemmet. Det var høyere forekomst av atferdsproblemer etter adopsjon. Atferden hadde forandret seg etter adopsjon, da det var noe mer atferdsproblemer og lavere hyppighet av normale atferder. Omplasseringskatter hadde mindre forekomst av atferdsproblemer enn huskatter som ikke hadde vært omplasserte. Kattene slo seg raskere til ro i det nye hjemmet, enn tidligere forsøk på omplasseringssenter. For de fleste atferdstrekkene opplevde eier at katten utførte dem i ønsket eller større grad. Tiden katten hadde vært hos eierne hadde ingen effekt på tilknytningsgraden mellom katt og eier. In this study a survey was conducted on two shelters in Oslo, Norway. The behaviour of 239 rehomed cats was registered on a questionnaire. The questionnaire included 12 questions regarding normal behaviour, problem behaviour and recommendations regarding the placement of a cat in the new home. The second questionnaire was sent out to the owners of the rehomed cats, about three months after the adoption, 65 responded. The questionnaire to the owner included 71 questions and was similar to the questions from the shelters, but more detailed about the environment, adoption and the relationship to the cat. The cats were neutered, or would it be if they were too young at the time of the adoption. 89 % of the respondents were female and the average age of the respondents were 40,8±9.9 years. 61,5 % of them lived in a highly populated area and 70,8 % of cats had the opportunity to walk freely outside, while only 12,3 % were kept as indoor cats. The remaining had a limited access to the outdoors. The average number of cats in each home was 1,42, and it took about six days before the cat settled down in their new home. 84,6 % had access to a litter box inside all year. On the shelters cats performed "positive" behavioral traits often or very often, like approaching strangers. The behavioural problems mostly occurred never or rarely, like aggressive biting towards humans. Scratching the inventory was the biggest behaviour problem. In the new home the behavioral traits were performed with high frequency, with the exception being when the cat approached children and unfamiliar cats, kept other cats away from the household and hunting. The most frequently executed problem behaviour was cats scratching the inventory, followed by fear and anxiety towards loud noises. Cats with free access to the outdoors showed problem behaviour to a greater extent than indoor cats. They would more often defecate outside the litter box. There were less behaviour problems in households with more than one cat, for example there were less aggressive biting and scratching towards humans. Owners, who had more than five years of experience with cats, observed less behaviour problems. When comparing the shelter cats from this study to cats from a previous study with a “normal” background, nine out of eleven behaviour problems were performed more often by the “normal” cats. They were more aggressive towards other cats and would more often urine mark inside. Scratching on the inventory was performed most by both of the groups.When asked about how much owners preferred certain behaviour traits in their cats, the response had a positive correlation with how often the cats executed the behaviour in half of the cases. About three months after the adoption 9,8 of 10 owners were satisfied or very satisfied with their cats. The owners scored their emotional bond to their cats strongly attached or very strongly attached in 8 out of 10 cases. The social bond between owner and cat had on a scale from 1 to 10, a mean of 8,43±1,48. When choosing a cat the owners put more weight on the cats’ personality, while appearance, background and recommendation from the shelter were of medium importance. Cats mainly chosen by their appearance, would have a less successful relationship with their owners. Behaviour traits were performed less in the cat’s new homes than at the shelters. Generally there were few behaviour problems at the shelters and in the new home. There was a higher frequency of behaviour problems after an adoption. The behaviour had changed after the adoption. The behaviour problems increased and the behaviour traits decreased. Cats from a shelter showed less of behaviour problems than cats with ‟normal” background. The cats settled faster in the new home, than former studies done at shelters. Time spent living together had no effect on the degree of attachment between cat and owner

Use of Liposomes to Study Cellular Osmosensors

When cells are exposed to changes in the osmotic pressure of the external medium, they respond with mechanisms of osmoregulation. An increase of the extracellular osmolality leads to the accumulation of internal solutes by biosynthesis or uptake. Particular bacterial transporters act as osmosensors and respond to increased osmotic pressure by catalyzing uptake of compatible solutes. The functions of osmosensing, osmoregulation , and solute transport of these transporters can be analyzed in molecular detail after solubilization, isolation, and reconstitution into phospholipid vesicles. Using this approach, intrinsic functions of osmosensing transporters are studied in a defined hydrophilic (access to both sides of the membrane) and hydrophobic surrounding (phospholipid membrane), and free of putative interacting cofactors and regulatory proteins

Efflux of compatible solutes in Corynebacterium glutamicum mediated by osmoregulated channel activity

Ruffert S, Lambert C, Peter H, Wendisch VF, Kramer R. Efflux of compatible solutes in Corynebacterium glutamicum mediated by osmoregulated channel activity. European Journal of Biochemistry. 1997;247(2):572-580.Bacteria respond to hypoosmotic stress by releasing low-molecular-mass solutes in older to maintain constant turgor pressure. We have studied the function of osmoregulated channel(s) in Corynebactium glutamicum which are responsible for efflux of various solutes upon sudden decrease in osmotic pressure. The channels preferentially mediated efflux of compatible solutes such as glycine betaine and proline. The release of molecules of similar size. e.g. glutamate or lysine, was restricted, ATP was completely retained even after severe osmotic shock. The cells maintained high cytoplasmic K+ and Na+ concentrations under hypoosmotic shock. Several results suggest that the solute efflux is mediated by a channel and not by a carrier. e.g. by reversal of the glycine betaine uptake systems of C. glutamicum,lf the release of glycine betaine and proline was extremely fast reaching an efflux rate of 6000 mu mol . min(-1). g dm(-1) or higher: the efflux was not significantly influenced by addition of external transport substrate, e.g. glycine betaine; in spite of an extremely high chemical gradient, no significant efflux under isoosmolar conditions was observed: efflux of solutes was unchanged after full uncoupling of membrane energetics by carbonylcyanide m-chlorophenylhydrazone (CCCP). These results indicate the presence of an osmoregulated channel in C. glutamicum similar to the mechanosensitive channel(s) of Escherichia coli. The activity of the channel did not depend on the growth conditions, but we observed a light regulation on the level of activity, i.e. the mechanosensitive channel behaved as a perfect osmometer. By monitoring release of glycine betaine under slow and continuous decrease of the external osmolality, we observed continous efflux without a stepwise release of solutes. This resulted in a significant steady-state decrease ase of the membrane potential