Transfer of reducing equivalents into mitochondria by the interconversions of proline and Δ1-pyrroline-5-carboxylate

Direct evidence is presented for a proline cycle using a cell-free experimental system which sequentially transfers ³H from [1-³H]glucose to NADP⁺ to Δ¹-pyrroline-5-carboxylate and yields [³H]proline. The formation of [³H]proline depends on the presence of NADP, Δ¹-pyrroline-5-carboxylate, and the enzymes glucose-6-phosphate dehydrogenase and Δ¹-pyrroline-5-carboxylate reductase. The production of [³H]proline from unlabeled proline in the presence of mitochondria provides direct evidence for one complete turn of a proline cycle which transfers reducing equivalents produced by glucose oxidation in the pentose pathway into mitochondria. In this cycle, proline is oxidized to Δ¹-pyrroline-5-carboxylate by mitochondrial proline oxidase. Δ¹-pyrroline-5-carboxylate is released from mitochondria and is recycled back to proline by Δ¹-pyrroline-5-carboxylate reductase with concomitant oxidation of NADPH. At the maximal rate observed, 60% of Δ¹-pyrroline-5-carboxylate produced is recycled back to proline. This cycle provides a mechanism for transferring reducing equivalents from NADPH into mitochondria and is linked to glucose oxidation in the pentose pathway by NADPH turnover.     

Influence of osmolarity and pH increase to achieve a reduction of monoclonal antibodies aggregates in a production process

Anti PSA monoclonal antibodies for diagnostic use were produced in an in vitro system. After purification using Protein G affinity chromatography a percentage of about 10% of antibody aggregates remained. The use of monoclonal antibodies containing aggregates as a capture antibody in a diagnostic kit reduces the performance of the test making it often unacceptable. The aggregates could be eliminated using gel filtration chromatography but, in that way, the final recovery of the whole production process was only about 50%. Aggregation is favoured when the working pH is near to the isoelectric point of the antibody. We varied the culture medium composition, modifying pH and osmolarity. We tested different values of pH and osmolarity: 7.1, 7.5, 8.0, 8.5 for pH, and 300, 340, 367, 395 mOsm/kg H2O for osmolarity. By modification of the cell culture medium we obtained a significant decrease of monoclonal antibody aggregates in the production cycle. In this way we achieved higher recovery rate and could avoid gel filtration polishing step. The experiments were performed in two stages: first in culture flasks changing one parameter in each experiment, and then in spinner bottle using the best conditions obtained in the first stage. During scale up we used the modifications achieved from the experiment showed in this paper in our production by hollow fibre bioreactor with positive results.     

HAM: A new epitope-tag for in vivo protein labeling

The ability to metabolically label proteins with ³⁵S-methionine is critical for the analysis of protein synthesis and turnover. Despite the importance of this approach, however, efficient labeling of proteins in vivo is often limited by a low number of available methionine residues, or by deleterious side-effects associated with protein overexpression. To overcome these limitations, we have created a methionine-rich variant of the widely used HA tag, called HAM, for use with ectopically expressed proteins. Here we describe the development of a series of vectors, and corresponding antisera, for the expression and detection of HAM-tagged proteins in mammalian cells. We show that the HAM tag dramatically improves the sensitivity of ³⁵S-methionine labeling, and permits the analysis of Myc oncoprotein turnover even when HAM-tagged Myc is expressed at levels comparable to that of the endogenous protein. Because of the improved sensitivity provided by the HAM tag, the vectors and antisera described here should be useful for the analysis of protein synthesis and destruction at physiological levels of protein expression.     

Untersuchungen zur Bestimmung des Geschlechts

Ohne Zusammenfassung     

Hermaphroditismus im Tierreich vom genetischen Standpunkt

Ohne Zusammenfassung     

Die Bedeutung des Y-Chromosoms im Tierreich

Ohne Zusammenfassung     

Vererbungsstudien

Ohne Zusammenfassung     

DasHelodea-Blatt als Testobjekt zum Studium der Wirkung von Schädlingsbekämpfungsmitteln

Zusammenfassung Die Praxis der Schädlingsbekämpfung wird auf die Möglichkeit hingewiesen, durch stärkere Berücksichtigung protoplasmatischer Gedankengänge und Methoden der Lösung ihrer Probleme näher zu kommen.Auf der anderen Seite eröffnen sich der Protoplasmaforschung durch Berücksichtigung der auftauchenden Fragestellungen neue Wege der Erkenntnis.An einigen praktischen Beispielen wird die Arbeitsmöglichkeit auf diesem Gebiete gezeigt. DasHelodea- Blatt erwies sich als ein ausgezeichnetes Versuchsobjekt zum Studium der Wirkung einzelner Gifte und, innerhalb gewisser Grenzen, als ein gutes Testobjekt zum Vergleich verschiedener oder verschieden angewandter Schädlingsbekämpfungsmittel.Es wird die Abtötungskurve desHelodea-Blattes für Nikotin aufgestellt.Nikotin kann in charakteristischer Weise auf die Wanderung der Piastiden Einfluß haben (Brückenstellung der Plastiden).Eine ähnliche Kurve wird für Schwefelkalk gegeben. Unter gewissen Bedingungen geht mit der Zellvergiftung eine Schwärzung des gesamten Plasmaapparates, vor allem zuerst der Plastiden, einher.Für die Vergiftung mit Kupferkalk gelten besondere Voraussetzungen, auch hier dürfte sich dasHelodea-Blatt als Versuchsobjekt bewähren.