Combining protein identification and quantification: C-terminal isotope-coded tagging using sulfanilic acid.

Two methods of differential isotopic coding of carboxylic groups have been developed to date. The first approach uses d0- or d3-methanol to convert carboxyl groups into the corresponding methyl esters. The second relies on the incorporation of two 18O atoms into the C-terminal carboxylic group during tryptic digestion of proteins in H(2)18O. However, both methods have limitations such as chromatographic separation of 1H and 2H derivatives or overlap of isotopic distributions of light and heavy forms due to small mass shifts. Here we present a new tagging approach based on the specific incorporation of sulfanilic acid into carboxylic groups. The reagent was synthesized in a heavy form (13C phenyl ring), showing no chromatographic shift and an optimal isotopic separation with a 6 Da mass shift. Moreover, sulfanilic acid allows for simplified fragmentation in matrix-assisted laser desorption/ionization (MALDI) due the charge fixation of the sulfonate group at the C-terminus of the peptide. The derivatization is simple, specific and minimizes the number of sample treatment steps that can strongly alter the sample composition. The quantification is reproducible within an order of magnitude and can be analyzed either by electrospray ionization (ESI) or MALDI. Finally, the method is able to specifically identify the C-terminal peptide of a protein by using GluC as the proteolytic enzyme.

Recueil de lettres et de pièces originales, et de copies de pièces indiquées comme telles dans le dépouillement qui suit.

Contient : 1 Lettre d'« ANTHOINE [duc DE LORRAINE]... à monseigneur d'Orval,... gouverneur et lieutenant general du roy en Champaigne... De Nancey, le penultiesme jour de may » ; 2 Lettre des « vicaire general de l'eveschié, clergé, loy, manans et habitans de la cité et ducé de Cambray... à... monseigneur l'admiral de France... De Cambray, ce XVIe jour de juing » ; 3 Lettre de « MARGUERITE [DE VALOIS, reine de Navarre]... à monsieur le chancellier d'Allençon... A Fontainebleau, le XXI jour de jullet » ; 4 Lettre de « MARGUERITE [DE VALOIS, reine de Navarre]... à monsieur le tresorier Robertet,... De Meaulx, ce penultiesme de septembre » ; 5 « Coppie d'unes lettres que ung secretaire du roy des Rommains... JEHAN LALLEMANT,... escripvoit à monseigneur Du Ru,... De Brucelles, samedi au soir VIme juillet » ; 6 Lettre de « JEHANNE [D'ALBRET, reine] de Navarre... à monsieur le chancelier d'Alençon » ; 7 Lettre de « CLAUDE DE LORRAINE [duc DE GUISE]... à monseigneur le tresorier Robertet,... Escript à Montereul, le XXe jour de janvier » ; 8 Lettre de « GALIOT [DE GENOUILLAC]... au roy... De Mouzon, ce VIe de juing » ; 9 Lettre des « gens des comptes du roy... à messire Florimont Robertet, chevalier, conseiller du roy et tresorier de France, baron d'Aluye,... Escript à Paris, le XIme jour de septembre » ; 10 Lettre de « GALIOT [DE GENOUILLAC]... au roy... Escript à Mouzon, le VIIIme jour de juing » ; 11 Lettre de « DINTEVILLE,... à monseigneur d'Alluye et de Bury, tresorier de France... A Dijon, ce Xe jour d'octobre » ; 12 Lettre d'ANNE DE « MONTMORENCY,... à monseigneur... de La Tremoille,... A Lucerne, le XXVIIIe jour de janvier » ; 13 Lettre de « M. SCURMAGATTA,... à madame la regente de France [Louise de Savoie]... De Toledo, ce second jour de juing, l'an 1525 » ; 14 Lettre de D'«HUMYERES,... à monsieur le tresorier Robertet,... A Peronne, ce XXIIe de septembre » ; 15 Lettre de « THELLIGNY,... à monseigneur le baron d'Alluye, tresorier de France... De Bordeaux, le XXIIe d'aoust » ; 16 Lettre de « SANTA COLOMA,... à monsieur le tresorier Robertet,... A Pampellonne, le XXIe jour de may » ; 17 Lettre, en latin, de « R. D. DETERBES » ; 18 Lettre de « D'ENTREMONS,... à monseigneur... Escript à Lenynville, le XVIIe jour de juillet » ; 19 Lettre de « NICOLAUS HAFFURER VON HEIDEG,... au roy... Escript à Fribourg, le premier jour de mars » ; 20 Lettre de « H. MOUILLY,... au roi... Escript à vostre ville françoyse de Grace, ce XXVIIme jour de janvier » ; 21 Copie d'une lettre des « advoyez de Lucerne TANMAN (sic), HERTTENSTEIN et ZUO KAES, avec le secretaire... A Lucerne, le IIIIe d'octobre anno [M.V.C.] XXI » ; 22 Lettre de « MEZIERES » et « MOY,... au roy... De Reyns, le VIIme septembre » ; 23 Lettre d'«OUDART DU BIES,... à monseigneur de La Fayette, seneschal et lieutenant pour le roy en Boullegnois... De Monstreuil, ce XIe de septembre » ; 24 Lettre de « MEZIERE,... à monseigneur d'Alluye,... A Ortal, ce XVIIIe jour de septembre » ; 25 Lettre de M. « DE HAPPLAINCOURT,... à monseigneur... le tresorier Robertet,... A Happlaincourt, le XIIIIe jour d'octobre » ; 26 Lettre de « L[OUIS] DE CANOSSAT DE BAIEUS,... à monseigneur... le tresorier Robertet,... De Senlis, le XIIIe d'octobre » ; 27 Lettre du « conte DE LUOPFFEN » et de « HANS VON BRANDES au roy... De Lisle, ce Xme jung » ; 28 Lettre du capitaine « LA CLAYETE,... au roy... A Luzy, le XIIe jour de jung » ; 29 Lettre de « ROBERT STUART [maréchal d'Aubigny]... au roy... A Dijon, ce XIIe jour de juing » ; 30 Dépêche, avec chiffre, de « F. LE ROUGE,... à monseigneur... le chancellier... De Venise, ce XIe de juillet » ; 31 Lettre de « PAULO CAMILLO TRIVULTIO,... au roy... Escript au camp à Suart, le VIe jour de septembre » ; 32 Lettre des « notaires et secretaires [du roi]... au roy... A Paris, le XIIIme jour d'aoust, l'an mil V.C.XXI » ; 33 « Double de lettres envoiées à monseigneur de Vendosme » ; 34 Lettre de « F. LE ROUGE,... à monseigneur le tresorier Robertet,... De Venise, ce XXIIe d'aoust » ; 35 Lettre de « MARC DE LA BAUME,... au roy... Escript à Lengres, ce VIIe de juing » ; 36 Pièce diplomatique en italien ; 37 Lettre, en latin, de « ULRICH, Hertzog ZU WIRTTENBERG [ULRIC, duc DE WURTEMBERG]... Francisco [I°], regi Francie... Datum ex Montepelligardo, XIIa octobris, anno M.D.XXI » ; 38 Lettre de « JEHAN PAPON,... à... monseigneur de Combronde,... lieutenant general en l'armée de Borgongne... Escript à Cuzet, ce dimenche au soir XIIIe jour d'avril » ; 39 Lettre sans signature ni adresse touchant les affaires de religion. Du « XXIIIe mars 1562 » ; 40 Lettre de « PIERRE DE LANOY, LA CLAYETE, FAYETE, SAINT ANDRE, CADOVAT,... au roy... Escript à Bayonne, le XVIIIe jour de juillet » ; 41 « Advertissement du camp de Francisque » ; 42 Lettre de « JEHAN PREVOST,... à monseigneur... d'Alluye,... A Paris, ce XXIXe janvier » ; 43 Lettre de « JOFFROY FEVYEY,... au roy... A Millan, le VII jour de juing » ; 44 Lettre de « M[ENAUD DE MARTRES DE SAINTE-COLOMBE], evesque de Tarbe... à monseigneur de Lautrec, conte de Foix et de Conmenge, gouverneur de Guyenne et lieutenant general du roy en Italie... A Millan, ce XXIIIe de juillet » ; 45 Lettre de « LOYS DE GENLY,... De Mouzon, ce VIIIe jour d'aoust » ; 46 « Double des lettres que aucuns bons personnaiges des ligues ont escriptes à monseigneur le grand maistre... Escript à Berne, le XXVme jour de janvier » ; 47 Lettre de « HIERONIMO TRIVULTIO » et « JO. FERMO TRIVULTIO,... au roy... A Parme, ce XXVe jour de jung » ; 48 Lettre de « JEHAN [STUART, duc d'Albany]... au roy... Escript à Sainct Saturonin, le Xe jour de jung » ; 49 Lettre de « CHARLES DE LUXEMBOURG, [Sr] DE BRYENNE,... au roy... Le IXe jour de septembre » ; 50 « Coppie de lettres escriptes par monseigneur D'ORVAL,... JEHAN D'ALEBRET,... à monsieur de Nansou,... A Maizieres, le XXe jour d'aoust » ; 51 « Double de la lettre escripte par le gouverneur de Genes... OCTAVIANO FREGOSO,... à monseigneur le marquis... De Genes, le IXme jour de juillet » ; 52 Lettre de « HANS VON BRANDES,... au roy... De Helicourt, ce VIIme juing » ; 53 Lettre de « SENTA COLOMA,... au roy... A Bayonne, ce XVIIIe jour de juillet » ; 54 « Coppie de la reponce en chifre faicte au cappitaine de Loges,... Donné à Callaix, ce XVIIIe d'aoust » ; 55 Lettre de « LA FAYETTE au roy... A Boullongne, ce samedi vigille de Nostre-Dame de septembre » ; 56 Lettre de « HE LOGES,... au roy... En vostre chasteau de Tournay, le XXIIIe jour de juillet » ; 57 Lettre de « LOYS DE ROUVILLE,... à... monseigneur le grant seneschal... De Harfleu, ce XXIIIe jour d'octobre » ; 58 Lettre de « CASSART,... à monseigneur le tresorier Robertet,....De Lion, ce Xe de jullet » ; 59 Lettre de « LOYS DE ROUVILLE,... au roy... De Harfleu, ce XXIIIme jour d'octobre » ; 60 « Double de lettres de OGIER DE SIGNY,... Escript à Mozon, le XIIIe jour d'aoust » ; 61 Lettre de M. « DE COULLIGNY » à son « frere monseigneur de Saligny,... Escript au Delost, ce jour de Pasques » ; 62 Lettre de M. « DE STAINVILLE,... à monseigneur d'Alye, conseiller du roy et tresorier de France... Escript à Nancy, le XXe jour d'aoust, à huict heures de soir » ; 63 Lettre de « GRAYAN DE GARRO,... au roy... A Come, ce dixiesme de jullet » ; 64 Lettre de « G. DE FURSTENBERG,... au roy... Escript à Heacourt, le VIIe de jung » ; 65 « Advertissement » sur la marche de l'armée impériale en 1521 ; 66 Lettre de « SENTA COLOMA,... Au camp lez Vienne, le VIme de juing » ; 67 Lettre de « G. DE FURSTENBERG,... au roy... Escript à Lile, le Xe de juing » ; 68 Lettre de « JEHAN D'ALHON,... au roy... Escript à Desize, le XIIme jour de may » ; 69 Copie d'une lettre à « monseigneur DE LOGES,... De Compiengne, ce XXVIme jour de novembre » ; 70 Copie d'une lettre à « messrs [de la ville de Tournay]... De Compiengne, ce XXVIme jour de novembre » ; 71 Lettre, en latin, de « SAXO,... dominis magno magistro admirato et magno scutifero ac Roberteto, secretario Francie... Ex Gullisa, XIIIa julii, hora medie noctis » ; 72 Lettre de « MARC DE LA BAUME,... au roy... Escript à Lengres, le cinquiesme jour de juing » ; 73 « Double de lettres de monseigneur DE BIERMONT,... De St-Amand, le cinquiesme juillet » ; 74 Lettre de « JEHAN [STUART, duc d'Albany]... à Madame... A Sainct Saturnin, le XVIIe aoust » ; 75 Lettre de « POYFOU,... au roy... A Reims, le XIIIIe jour de septembre » ; 76 « Coppie des lettres envoyées à monsieur de La Tremoille par monsieur DES REAUX,... De Losanne, ce XIIe jour d'aoust » ; 77 Lettre de « HE. LOGES » à « monseigneur de Chastillon, mareschal de France » ; 78 Lettre de « GUILLAUME [PETIT], evesque de Troyes, confesseur du roy... à... monseigneur le tresorier Robertet,... A Sainct Seine, ce XXVe de juillet » ; 79 Lettre de « JEHENNE D'ESTOUTEVILLE,... à monseigneur le tresorier Robertet,... De Neufviz, ce XVe septembre » ; 80 Lettre de « l'advoyer, petit et grand conseil de Lucerne... à... monseigneur de Lamet, ambassadeur du roy tres chrestien... Donné à Lucerne, le IIe de septembre, anno etc. XXI° » ; 81 « Double des lettres escriptes par les ambassadeurs du roy estans à Calais aux officiers du roy à Monstreul. Du XVIIIme jour d'aoust » ; 82 « Double de la lettre escripte par Don FRANÇOYS DE BEAUMONT à monseigneur d'Estissac,... De Epille, le XXXe de juillet » ; 83 « Coppie des lettres envoyéez à monseigneur de La Tremoille par le bailly de Chaalon et l'un des commissaires sur la conduite des Souysses... De Chaalon, ce XVe d'aoust » ; 84 Lettre d'un gouverneur du Havre « au roy [François Ier]... A vostre ville françoyse de Grace, ce XXVIme jour de janvier » ; 85 Lettre de « F. LE ROUGE » au roi. « De Venise, ce XIe de juillet » ; 86 Lettre de « CLEMENS CHAMPION,... à monseigneur... le chancellier de France ou à monseigneur de St Marsault, et en leur absence à messrs Robertet et Villeroy,... De Millan, a XX de augst mil cinqC. XXI » ; 87 « Lettres du connestable de Castele, du XXVe de may, au marquis de Falses » ; 88 « Lettres dudict connestable, du XXVe may, à Donanto, filz du marquis de Falses » ; 89 « Advertissemens de Castille envoyez au marquis de Falses par ledict connestable » ; 90 Lettre de « FRANCYSQUE,... au roy... De Genes, ce XVIIme de janvyer » ; 91 Lettre de « RAOUL HURAULT,... au roy... Escript à Athigny, ce XIme jour de juillet » ; 92 Lettre de « STAINVILLE,... à monseigneur de Villeroy, conseilier du roy et secretaire de sez finances... De Nancey, le XXe jour d'aoust, à huict heures du soir » ; 93 « Lettres dechiffrées du cappitaine LOGES,... Au Chasteau de Tournay, le unziesme jour d'aoust » ; 94 Copie d'une lettre au roi Françoys Ier, touchant l'entretien des troupes suisses ; 95 « Double des lettres que monseigneur D'ASPARROZ escript au roy... Au camp de Thiebes, le XXIXe jour de juing » ; 96 Lettre de « LA FAYETE,... au roy... A Boullongne, ce IXe jour de juing » ; 97 Lettre de « BREZE,... au roy... De vostre maison d'Ennet, ce dernier jour de novembre » ; 98 Lettre de « CAUSSIEUVILLE » au roi. « Du camp, se XIIe d'octobre » ; 99 Lettre de « G. DE LA CHASTRE » au roi. « Andelot, ce Xe jour de juing » ; 100 Lettre close « de par l'empereur... CHARLES [QUINT]... Donné en nostre ville de Gand, le XXIIIe jour de juillet XV.C.XXI » ; 101 Copie d'une lettre de « LE CHARRON,... au roy... En Alexandrie, ce XXIIIe de novembre » ; 102 Lettre de « FRANÇOYS DE SILLY,... à monseigneur... d'Artel,... ce jour Nostre Dame, VIIIe jour de septembre » ; 103 Lettre des « commissaires des estatz de Normandie... à monseigneur d'Alluye,... tresorier de France... Escript à Rouen, le XIIIe jour d'aoust » ; 104 Lettre de « G. DE LA CHASTRE,... à monseigneur d'Aluye,... A Troyes, vandredi XIIe » ; 105 Lettre de « RAOUL HURAULT,... au roy... Escript à Athigny, ce XXIIIe de juillet » ; 106 Lettre de « LA CLAYETE,... à monseigneur... le tresorier Robertet,... Escript à Bayonne, le XIXe jour d'aoust » ; 107 Lettre de « GUY DE LAVAL,... au roy... Escrypt au Havre de Blahouet, le XXVIe jour d'aoust » ; 108 Lettre des « commissaires des estatz de Normandie... au roy... Escript en ceste vostre bonne ville de Rouen, le XIIIme d'aoust » ; 109 Lettre de « THOMAS BOHIER,... au roy... Escript à Yrynorance pres Fontarabye, le Ve jour d'octobre » ; 110 « Lectres de ceulx de la ville de Steille au roy de Navarre, dattez du XXVme de juing ». Copie ; 111 « Lectres du XXVIme de juing, escriptes à Pampellone et adressez au roy de Navarre ». Copie ; 112 Lettre de « THOMAS BOHIER,... au roy... Escript à Handaye pres Fontarrabie, le XXIIIe jour d'octobre » ; 113 Lettre de « HENRY BOHIER,... au roy... Escript à Lyon, le VIme jour d'octobre » ; 114 « Translat de la lettre que messrs de Berne ont escripte aux Suysses qui sont au service du roy... Donné em haste, le jeudi devant Sainct Bartholomieu » ; 115 Lettre de MM. « DEMAZIS, JEHAN SERILHAN,... au roy... De vostre ville de Montpellier, le XXIe jour de septembre » ; 116 Lettre de « LE VISTE et P. LEGENDRE,... au roy... A Paris, le XVme aoust » ; 117 Lettre de « P. DE GAURE,... à messrs les quattre consaulx de la ville et cité de Tournay... Escript à Gand, ce XXIe de juillet »

Participaci??n y gesti??n democr??tica en los centros, garant??a de una escuela p??blica de calidad

Monogr??fico con el t??tulo: 'Los consejos escolares, cauces de participaci??n de la comunidad educativa'. Resumen basado en el de la publicaci??n

Alocação de biomassa e nutrientes em Heteranthera reniformis sob o efeito de N, P e K

Este trabalho objetivou estudar a distribuição de biomassa e nutrientes entre os componentes vegetativos de Heteranthera reniformis sob o efeito de diferentes concentrações de N, P e K. As plantas foram cultivadas em vasos plásticos preenchidos com pedra rolada, em soluções nutritivas a 80% da concentração original de Sarruge, sendo esta a solução-base. O delineamento experimental utilizado foi o inteiramente casualizado, com quatro níveis (0, 25, 50 e 75% da solução-base) avaliados individualmente em N, P e K, além da testemunha (100% da solução-base), com quatro repetições no período de 35 dias. As maiores áreas foliares ocorreram em soluções com 75% de P e 50% de N e K. A solução com 50% de N proporcionou a maior biomassa seca de plantas. A contribuição do caule na alocação de recursos apresentou resultados que variaram entre 55,5 e 74,5% em relação à matéria seca total referente ao nível 0 e 75% de N; a contribuição de raízes reduziu com o aumento da concentração de N, bem como de P e K. As maiores contribuições de raízes ocorreram nas ausências de N (26,8%) e P (19,2%), os quais foram maiores em relação aos seus respectivos níveis mais concentrados. Os teores de N, P e K corresponderam a uma ordem crescente em folha, caule e raízes, em que os maiores teores foram obtidos nos níveis de 50 a 125 mg L-1 de N, 5 a 15 mg L-1 de P e 100 a 150 mg L- 1 de K.

Neovascularization after ischemic injury: evaluation with 99mTc-HYNIC-RGD

Purpose: Angiogenesis involves many mediators including integrins, and the tripeptide RGD is a target amino acid recognition sequence for many of them. Hindlimb ischemia is a simple and convenient animal model however standardization of the injection procedures in the devascularized and control limb is lacking, thus rendering difficult the interpretation of results. The aim of this investigations was to evaluate neovascularization in a hindlimb murine model by means of 99mTc-HYNIC-ß-Ala-RGD. Methods: 99mTc-HYNIC-RGD analog was prepared using coligands. Ischemia was induced in Wistar rats by double- ligation of the common femoral artery. Radiolabeled RGD was injected after 2h, as well as 1, 3, 5, 7, 10 and 14 days. Uptake was evaluated by planar imaging and biodistribution studies. Results: The highest ratio between ischemia and control was achieved at the 7th day (2.62 ± 0.95), with substantial decrease by the 14th day. For pertechnetate the 7th day ratio was 0.87 ± 0.23. Scintigraphic image confirmed different uptakes. Conclusion: 99mTc-HYNIC-RGD analog concentrated in ischemic tissue by the time of widespread angiogenesis and pertechnetate confirmed reduction in blood flow. In this sense, the protocol can be recommended for ischemic models.

Einfluss der Modulation von P-Glykoprotein (P-gp) auf die Verfügbarkeit und Wirksamkeit der pharmakologischen Behandlung psychiatrischer Erkrankungen im Tiermodell

P-Glykoprotein (P-gp) ist ein ATP-verbrauchender Transporter, der in Organschranken exprimiert wird, um Fremdstoffe auszuschleusen, darunter auch Psychopharmaka. Im Rahmen dieser Arbeit wurde im Tiermodell der Maus untersucht, welche pharmakokinetischen und pharmakodynamischen Konsequenzen sich bei Verabreichung von Risperidon als P-gp Modellsubstrat ergeben, wenn die Expression von P-gp induziert wird. Als potenzielle Induktoren wurden Dexamethason, Rifampicin, Quercetin, 5-Pregnen-3ß-ol-20-on-16α-Carbonitril (PCN) und Acitretin geprüft. Es konnte gezeigt werden, dass alle Substanzen die Verteilung von Risperidon und seinem aktiven Metaboliten 9-Hydroxyrisperidon beeinflussten. Während sich für Quercetin und Acitretin leichte P-gp inhibitorische Eigenschaften ergaben, die an Hand von erhöhten Konzentrationen von Risperidon und 9-Hydroxyrisperidon gezeigt werden konnten, führten die bekannten P-gp Induktoren Rifampicin, Dexamethason und PCN zu verringerten Konzentrationen im Vergleich zur Kontrollgruppe. Durch Western Blot Untersuchungen wurde bestätigt, dass die Induktoren die P-gp Expression im Hirngewebe tendenziell steigerten. Dies sprach dafür, dass bei Verabreichung einer Komedikation, die P-gp induziert, mit einer veränderten Verteilung von P-gp Substraten zu rechnen ist. Darüber hinaus konnte nachgewiesen werden, dass durch eine Hemmung bzw. Induktion von P-gp nicht nur die Pharmakokinetik, sondern auch die Pharmakodynamik von Risperidon und 9-Hydroxyrisperidon verändert wird. Dies wurde durch verhaltenspharmakologische Untersuchungen gezeigt. Durch Risperidon induzierte motorische Effekte auf dem RotaRod waren nach Induktion von P-gp abgeschwächt. Dies zeigte sich auch für Haloperidol, welches kein Substrat ist. Da P-gp abhängige Effekte in diesem Fall keine bedeutende Rolle spielen, ist davon auszugehen, dass neben der Induktion von P-gp an der Blut-Hirn Schranke auch andere Mechanismen wie z.B. eine Induktion von Enzymen der CYP-Familie an den beobachteten Effekten beteiligt sind. Bei Untersuchungen von kognitiven Leistungen in der Barnes Maze konnte gezeigt werden, dass Haloperidol im Gegensatz zu Risperidon das Lernverhalten negativ beeinflussen kann. Eine P-gp Induktion schien jedoch keinen deutlichen Einfluss auf das Lernverhalten unter Antipsychotika-Gabe zu haben und sprach vielmehr für substanzabhängige Effekte der einzelnen Antipsychotika bzw. P-gp Modulatoren. Zusatzuntersuchungen zur Hirngängigkeit von Acitretin, einem synthetischen Retinoid, welches derzeit als potenzielles Antidementivum geprüft wird, konnten belegen, dass es die Blut-Hirn Schranke überwindet. Bereits 1h nach Injektion war Acitretin in hoher Konzentration im Gehirn nachweisbar. Durch die Analyse zur Verteilung von Acitretin in Hirngewebe und Serum von P-gp Wildtyp und P-gp doppel knockout Mäusen konnte belegt werden, dass Acitretin nicht P-gp abhängig transportiert wird. Die Daten insgesamt betrachtet, lassen den Schluss zu, dass durch Verabreichung von Medikamenten, die P-gp Modulatoren sind, bei Antipsychotika mit pharmakokinetischen Interaktionen zu rechnen ist, welche die Wirksamkeit der Medikamente einschränken können.

Stable isotope stratigraphy of ODP Site 167-1014

Late Quaternary oxygen (d18O) and carbon (d13C) isotopic records for the benthic foraminifer Uvigerina and the planktonic foraminifer Globigerina bulloides are presented for the upper 20 meters composite depth sediment sequence of Ocean Drilling Program Site 1014, Tanner Basin, in the outer California Borderland province. The benthic oxygen isotopic record documents a continuous >160-k.y. sequence from marine isotope Stage (MIS) 6 to the present day. The record closely resembles other late Quaternary North Pacific benthic isotope records, as well as the well-dated deep-sea sequence (SPECMAP), and thus provides a detailed chronologic framework. Site 1014 provides a useful record of the California response to climate change as it enters the southern California Border-land. Sedimentation rates are relatively constant and high (~11.5 cm/k.y. ). The planktonic foraminiferal record is well pre-served except during marine isotope Substages 5b and 5d, when normally high G. bulloides abundance is strongly diminished as a result of dissolution. The planktonic oxygen isotopic shift of ~3 per mil between the last glacial maximum and the Holocene suggests a surface water temperature shift of <7°C, similar to estimates from Hole 893A (Leg 146) to the north. Unlike Santa Barbara Basin, G. bulloides d18O values during the last interglacial (MIS 5) at Site 1014 were significantly higher than during the Holocene. In particular, marine isotope Substage 5e (Eemian) was ~0.8 per mil higher. This is unlikely to reflect a cooler Eemian but is instead the result of preferential dissolution of thin-shelled (low d18O) specimens during this interval. In this mid-depth basin, a large benthic d18O shift during Termination I suggests dramatic temperature and salinity changes in response to switches in the source of North Pacific Intermediate Water. Although d13C values of the planktonic foraminifer G. bulloides are in disequilibria with seawater and hence interpretations are limited, the G. bulloides record exhibits several negative d13C excursions found at other sites in the region (Sites 1017 and 893). This indicates a response of G. bulloides d13C to regional surface water processes along the southern California margin. A general increase in benthic carbon isotopic values (-1.75 per mil to -0.75 per mil) in Tanner Basin during the last 200 k.y. is overprinted with smaller fluctuations correlated with climate change. The coolest intervals during the last glacial maximum (MISs 2 and 4) exhibit lower benthic d13C values, which correlate with global 13C shifts. The opposite relationship is exhibited during the last interglacial before 85 ka, when lower benthic d13C values are associated with warmer intervals (marine isotope Substages 5c and 5e) of the last interglacial. These time intervals were also marked by decreased intermediate water ventilation. Increased dissolution and organic accumulation during Substages 5b and 5d are anticorrelated with the benthic d13C record. These results suggest that a delicate balance in intermediate water d13C has existed between the relative influences of global 13C and regional ventilation changes at the 1165-m water depth of Site 1014.

R.P.F. Philippi Diez lusitani, Ordinis Minorum ... Quadruplicium concionum, quae quotidie à D[omi]nica in Septuagesima vsq[ue] ad gloriosam Domini Resurrectionem in sancta Ecclesia habentur : tomi primi prima et secunda pars.

Mode of access: Internet.

Editorial overview:new protein production tools for structural biology

Full text: The idea of producing proteins from recombinant DNA hatched almost half a century ago. In his PhD thesis, Peter Lobban foresaw the prospect of inserting foreign DNA (from any source, including mammalian cells) into the genome of a λ phage in order to detect and recover protein products from Escherichia coli [ 1 and 2]. Only a few years later, in 1977, Herbert Boyer and his colleagues succeeded in the first ever expression of a peptide-coding gene in E. coli — they produced recombinant somatostatin [ 3] followed shortly after by human insulin. The field has advanced enormously since those early days and today recombinant proteins have become indispensable in advancing research and development in all fields of the life sciences. Structural biology, in particular, has benefitted tremendously from recombinant protein biotechnology, and an overwhelming proportion of the entries in the Protein Data Bank (PDB) are based on heterologously expressed proteins. Nonetheless, synthesizing, purifying and stabilizing recombinant proteins can still be thoroughly challenging. For example, the soluble proteome is organized to a large part into multicomponent complexes (in humans often comprising ten or more subunits), posing critical challenges for recombinant production. A third of all proteins in cells are located in the membrane, and pose special challenges that require a more bespoke approach. Recent advances may now mean that even these most recalcitrant of proteins could become tenable structural biology targets on a more routine basis. In this special issue, we examine progress in key areas that suggests this is indeed the case. Our first contribution examines the importance of understanding quality control in the host cell during recombinant protein production, and pays particular attention to the synthesis of recombinant membrane proteins. A major challenge faced by any host cell factory is the balance it must strike between its own requirements for growth and the fact that its cellular machinery has essentially been hijacked by an expression construct. In this context, Bill and von der Haar examine emerging insights into the role of the dependent pathways of translation and protein folding in defining high-yielding recombinant membrane protein production experiments for the common prokaryotic and eukaryotic expression hosts. Rather than acting as isolated entities, many membrane proteins form complexes to carry out their functions. To understand their biological mechanisms, it is essential to study the molecular structure of the intact membrane protein assemblies. Recombinant production of membrane protein complexes is still a formidable, at times insurmountable, challenge. In these cases, extraction from natural sources is the only option to prepare samples for structural and functional studies. Zorman and co-workers, in our second contribution, provide an overview of recent advances in the production of multi-subunit membrane protein complexes and highlight recent achievements in membrane protein structural research brought about by state-of-the-art near-atomic resolution cryo-electron microscopy techniques. E. coli has been the dominant host cell for recombinant protein production. Nonetheless, eukaryotic expression systems, including yeasts, insect cells and mammalian cells, are increasingly gaining prominence in the field. The yeast species Pichia pastoris, is a well-established recombinant expression system for a number of applications, including the production of a range of different membrane proteins. Byrne reviews high-resolution structures that have been determined using this methylotroph as an expression host. Although it is not yet clear why P. pastoris is suited to producing such a wide range of membrane proteins, its ease of use and the availability of diverse tools that can be readily implemented in standard bioscience laboratories mean that it is likely to become an increasingly popular option in structural biology pipelines. The contribution by Columbus concludes the membrane protein section of this volume. In her overview of post-expression strategies, Columbus surveys the four most common biochemical approaches for the structural investigation of membrane proteins. Limited proteolysis has successfully aided structure determination of membrane proteins in many cases. Deglycosylation of membrane proteins following production and purification analysis has also facilitated membrane protein structure analysis. Moreover, chemical modifications, such as lysine methylation and cysteine alkylation, have proven their worth to facilitate crystallization of membrane proteins, as well as NMR investigations of membrane protein conformational sampling. Together these approaches have greatly facilitated the structure determination of more than 40 membrane proteins to date. It may be an advantage to produce a target protein in mammalian cells, especially if authentic post-translational modifications such as glycosylation are required for proper activity. Chinese Hamster Ovary (CHO) cells and Human Embryonic Kidney (HEK) 293 cell lines have emerged as excellent hosts for heterologous production. The generation of stable cell-lines is often an aspiration for synthesizing proteins expressed in mammalian cells, in particular if high volumetric yields are to be achieved. In his report, Buessow surveys recent structures of proteins produced using stable mammalian cells and summarizes both well-established and novel approaches to facilitate stable cell-line generation for structural biology applications. The ambition of many biologists is to observe a protein's structure in the native environment of the cell itself. Until recently, this seemed to be more of a dream than a reality. Advances in nuclear magnetic resonance (NMR) spectroscopy techniques, however, have now made possible the observation of mechanistic events at the molecular level of protein structure. Smith and colleagues, in an exciting contribution, review emerging ‘in-cell NMR’ techniques that demonstrate the potential to monitor biological activities by NMR in real time in native physiological environments. A current drawback of NMR as a structure determination tool derives from size limitations of the molecule under investigation and the structures of large proteins and their complexes are therefore typically intractable by NMR. A solution to this challenge is the use of selective isotope labeling of the target protein, which results in a marked reduction of the complexity of NMR spectra and allows dynamic processes even in very large proteins and even ribosomes to be investigated. Kerfah and co-workers introduce methyl-specific isotopic labeling as a molecular tool-box, and review its applications to the solution NMR analysis of large proteins. Tyagi and Lemke next examine single-molecule FRET and crosslinking following the co-translational incorporation of non-canonical amino acids (ncAAs); the goal here is to move beyond static snap-shots of proteins and their complexes and to observe them as dynamic entities. The encoding of ncAAs through codon-suppression technology allows biomolecules to be investigated with diverse structural biology methods. In their article, Tyagi and Lemke discuss these approaches and speculate on the design of improved host organisms for ‘integrative structural biology research’. Our volume concludes with two contributions that resolve particular bottlenecks in the protein structure determination pipeline. The contribution by Crepin and co-workers introduces the concept of polyproteins in contemporary structural biology. Polyproteins are widespread in nature. They represent long polypeptide chains in which individual smaller proteins with different biological function are covalently linked together. Highly specific proteases then tailor the polyprotein into its constituent proteins. Many viruses use polyproteins as a means of organizing their proteome. The concept of polyproteins has now been exploited successfully to produce hitherto inaccessible recombinant protein complexes. For instance, by means of a self-processing synthetic polyprotein, the influenza polymerase, a high-value drug target that had remained elusive for decades, has been produced, and its high-resolution structure determined. In the contribution by Desmyter and co-workers, a further, often imposing, bottleneck in high-resolution protein structure determination is addressed: The requirement to form stable three-dimensional crystal lattices that diffract incident X-ray radiation to high resolution. Nanobodies have proven to be uniquely useful as crystallization chaperones, to coax challenging targets into suitable crystal lattices. Desmyter and co-workers review the generation of nanobodies by immunization, and highlight the application of this powerful technology to the crystallography of important protein specimens including G protein-coupled receptors (GPCRs). Recombinant protein production has come a long way since Peter Lobban's hypothesis in the late 1960s, with recombinant proteins now a dominant force in structural biology. The contributions in this volume showcase an impressive array of inventive approaches that are being developed and implemented, ever increasing the scope of recombinant technology to facilitate the determination of elusive protein structures. Powerful new methods from synthetic biology are further accelerating progress. Structure determination is now reaching into the living cell with the ultimate goal of observing functional molecular architectures in action in their native physiological environment. We anticipate that even the most challenging protein assemblies will be tackled by recombinant technology in the near future.

Age models, accumulation rates, and grain size distributions of sediment cores from the northern subtropical Atlantic

The terrigenous sediment proportion of the deep sea sediments from off Northwest Africa has been studied in order to distinguish between the aeolian and the fluvial sediment supply. The present and fossil Saharan dust trajectories were recognized from the distribution patterns of the aeolian sediment. The following timeslices have been investigated: Present, 6,000, 12,000 and 18,000 y. B. P. Furthermore, the quantity of dust deposited off the Saharan coast has been estimated. For this purpose, 80 surface sediment samples and 34 sediment cores have been analysed. The stratigraphy of the cores has been achieved from oxygen isotopic curves, 14C-dating, foraminiferal transfer temperatures, and carbonate contents. Silt sized biogenic opal generally accounts for less than 2 % of the total insoluble sediment proportion. Only under productive upwelling waters and off river mouths, the opal proportion exceeds 2 % significantly. The modern terrigenous sediment from off the Saharan coast is generally characterized by intensely stained quartz grains. They indicate an origin from southern Saharan and Sahelian laterites, and a zonal aeolian transport in midtropospheric levels, between 1.5 an 5.5 km, by 'Harmattan' Winds. The dust particles follow large outbreaks of Saharan air across the African coast between 15° and 21° N. Their trajectories are centered at about 18° N and continue further into a clockwise gyre situated south of the Canary Islands. This course is indicated by a sickle-shaped tongue of coarser grain sizes in the deep-sea sediment. Such loess-sized terrigenous particles only settle within a zone extending to 700 km offshore. Fine silt and clay sized particles, with grain sizes smaller than 10- 15 µm, drift still further west and can be traced up to more than 4,000 km distance from their source areas. Additional terrigenous silt which is poor in stained quartz occurs within a narrow zone off the western Sahara between 20° and 27° N only. It depicts the present dust supply by the trade winds close to the surface. The dust load originates from the northwestern Sahara, the Atlas Mountains and coastal areas, which contain a particularly low amount of stained quartz. The distribution pattern of these pale quartz sediments reveals a SSW-dispersal of dust being consistent with the present trade wind direction from the NNE. In comparison to the sediments from off the Sahara and the deeper subtropical Atlantic, the sediments off river mouths, in particular off the Senegal river, are characterized by an additional input of fine grained terrigenous particles (< 6 µm). This is due to fluvial suspension load. The fluvial discharge leads to a relative excess of fine grained particles and is observed in a correlation diagram of the modal grain sizes of terrigenous silt with the proportion of fine fraction (< 6 µm). The aeolian sediment contribution by the Harmattan Winds strongly decreased during the Climatic Optimum at 6,000 y. B. P. The dust discharge of the trade winds is hardly detectable in the deep-sea sediments. This probably indicates a weakened atmospheric circulation. In contrast, the fluvial sediment supply reached a maximum, and can be traced to beyond Cape Blanc. Thus, the Saharan climate was more humid at 6,000 y B. P. A latitudinal shift of the Harmattan driven dust outbreaks cannot be observed. Also during the Glacial, 18,000 y. B. P., Harmattan dust transport crossed the African coast at latitudes of 15°-20° N. Its sediment load increased intensively, and markedly coarser grains spread further into the Atlantic Ocean. An expanded zone of pale-quart sediments indicates an enhanced dust supply by the trade winds blowing from the NE. No synglacial fluvial sediment contribution can be recognized between 12° and 30° N. This indicates a dry glacial climate and a strengthened stmospheric circulation over the Sahelian and Saharan region. The climatic transition pahes, at 12, 000 y. B. P., between the last Glacial and the Intergalcial, which is compareable to the Alerod in Europe, is characterized by an intermediate supply of terrigenous particles. The Harmattan dust transport wa weaker than during the Glacial. The northeasterly trade winds were still intensive. River supply reached a first postglacial maximum seaward of the Senegal river mouth. This indicates increasing humidity over the southern Sahara and a weaker atmospheric circulation as compared to the glacial. The accumulation rates of the terrigenous silt proportion (> 6 µm) decrcase exponentially with increasing distance from the Saharan coast. Those of the terrigenous fine fraction (< 6 µm) follow the same trend and show almost similar gradients. Accordingly, also the terrigenous fine fraction is believed to result predominantly from aeolian transport. In the Atlantic deep-sea sediments, the annual terrigenous sediment accumulation has fluctuated, from about 60 million tons p. a. during the Late Glacial (13,500-18,000 y. B. P, aeolian supply only) to about 33 million tons p. a. during the Holocene Climatic Optimum (6,000-9,000 y. B. P, mainly fluvial supply), when the river supply has reached a maximum, and to about 45 million tons p. a. during the last 4,000 years B. P. (fluvial supply only south of 18° N).

Chemical and isotopic compositions of water, particulates, plankton, gases, sediments, and minerals from the Kara Sea and lower Ob and Yenisei Rivers

The Kara Sea is an area uniquely suitable for studying processes in the river-sea system. This is a shallow sea, into which two great Siberian rivers, Yenisei and Ob, flow. From 1995 to 2003, the sea was studied by six international expeditions onboard the R/V Akademik Boris Petrov. This publication summarizes the results obtained, within the framework of this project, at the Vernadsky Institute of Geochemistry and Analytical Chemistry, Russian Academy of Sciences. Various hydrogeochemical parameters, concentrations and isotopic composition of organic and carbonate carbon of the sediments, plankton, particulate organic matter, hydrocarbons, and dissolved CO2 were examined throughout the whole sea area at more than 200 sites. The d13C varies from -22 and -24 per mil where Atlantic waters enter the Kara Sea and in the north-eastern part of the water area to -27 per mil in the Yenisei and Ob estuaries. The value of d13C of the plankton is only weakly correlated with the d13C of the organic matter from the sediments and is lower by as much as 3-4 per mil. The paper presents the results obtained from a number of meridional river-sea profiles. It was determined from the relations between the isotopic compositions of plankton and particulate matter that the river waters carry material consisting of 70% detrital-humus matter and 30% planktonogenic material in the river part, and the material contained in the offshore waters consists of 30% terrigenous components, with the contribution of bioproducers amounting to 70%. The carbon isotopic composition of the plankton ranges from -29 to -35 per mil in the riverine part, from -28 to -27 per mil in the estuaries, and from -27.0 to -25 per mil in the marine part. The relative lightness of the carbon isotopic composition of plankton in Arctic waters is explained by the temperature effect, elevated CO2 concentrations, and long-distance CO2 supply to the sea with river waters. The data obtained on the isotopic composition of CO2 in the surface waters of the Kara Sea were used to map the distribution of d13C. The complex of hydrocarbon gases extracted from the waters included methane, C2-C5, and unsaturated C2=-C4= hydrocarbons, for which variations in the concentrations in the waters were studied along river-estuary-sea profiles. The geochemistry of hydrocarbon gases in surface fresh waters is characterized by comparable concentrations of methane (0.3-5 µl/l) and heavier hydrocarbons, including unsaturated ones. Microbiological methane with d13C from -105 to -90 per mil first occurs in the sediments at depths of 40-200 cm. The sediments practically everywhere display traces of methane oxidation in the form of a shift of the d13C of methane toward higher values and the occurrence of autogenic carbonate material, including ikaite, enriched in the light isotope. Ikaite (d13C from -25 to -60 per mil) was found and examined in several profiles. The redox conditions in the sediments varied from normal in the southern part of the sea to highly oxidized along the Novaya Zemlya Trough. Vertical sections through the sediments of the latter exemplify the complete suppression of the biochemical activity of microorganisms. Our data provide insight into the biogeochemistry of the Kara Sea and make it possible to specify the background values needed for ecological control during the future exploration operations and extraction of hydrocarbons in the Kara Sea.

Occurrence and Function of Hoogsteen Base Pairs in Nucleic Acids

Nucleic acids (DNA and RNA) play essential roles in the central dogma of biology for the storage and transfer of genetic information. The unique chemical and conformational structures of nucleic acids – the double helix composed of complementary Watson-Crick base pairs, provide the structural basis to carry out their biological functions. DNA double helix can dynamically accommodate Watson-Crick and Hoogsteen base-pairing, in which the purine base is flipped by ~180° degrees to adopt syn rather than anti conformation as in Watson-Crick base pairs. There is growing evidence that Hoogsteen base pairs play important roles in DNA replication, recognition, damage or mispair accommodation and repair. Here, we constructed a database for existing Hoogsteen base pairs in DNA duplexes by a structure-based survey from the Protein Data Bank, and structural analyses based on the resulted Hoogsteen structures revealed that Hoogsteen base pairs occur in a wide variety of biological contexts and can induce DNA kinking towards the major groove. As there were documented difficulties in modeling Hoogsteen or Watson-Crick by crystallography, we collaborated with the Richardsons’ lab and identified potential Hoogsteen base pairs that were mis-modeled as Watson-Crick base pairs which suggested that Hoogsteen can be more prevalent than it was thought to be. We developed solution NMR method combined with the site-specific isotope labeling to characterize the formation of, or conformational exchange with Hoogsteen base pairs in large DNA-protein complexes under solution conditions, in the absence of the crystal packing force. We showed that there are enhanced chemical exchange, potentially between Watson-Crick and Hoogsteen, at a sharp kink site in the complex formed by DNA and the Integration Host Factor protein. In stark contrast to B-form DNA, we found that Hoogsteen base pairs are strongly disfavored in A-form RNA duplex. Chemical modifications N1-methyl adenosine and N1-methyl guanosine that block Watson-Crick base-pairing, can be absorbed as Hoogsteen base pairs in DNA, but rather potently destabilized A-form RNA and caused helix melting. The intrinsic instability of Hoogsteen base pairs in A-form RNA endows the N1-methylation as a functioning post-transcriptional modification that was known to facilitate RNA folding, translation and potentially play roles in the epitranscriptome. On the other hand, the dynamic property of DNA that can accommodate Hoogsteen base pairs could be critical to maintaining the genome stability.