We have isolated a gene (WS5) that is specifically expressed at the mRNA and protein level in avian fibroblasts transformed by the v-myc oncogene of avian acute leukemia virus MC29. In a conditional cell transformation system, WS5 gene expression was tightly correlated with v-myc activation. The WS5 gene contains 11 exons, encoding a 733-amino acid protein with a transmembrane region and a polycystic kidney disease (PKD) domain. Near the transcriptional start site, the WS5 promoter contains a cluster of four binding sites for the Myc-Max complex and a binding site for transcription factor C/EBPa. Electrophoretic mobility shift assays and chromatin immunoprecipitation showed that Myc, Max and C/EBPa bind specifically to these sites. Functional promoter analyses revealed that both the Myc-binding site cluster and the C/EBPa-binding site are essential for strong transcriptional activation, and that Myc and C/EBPa synergistically activate the WS5 promoter. Ectopic expression of WS5 led to cell transformation documented by anchorage-independent growth. The human melanoma antigen Pmel17, a type I transmembrane glycoprotein, is the mammalian protein with the highest amino acid sequence identity (38%) to WS5. The Pmel17 gene is regulated by the MITF protein, a bHLHZip transcription factor with DNA binding specificities similar to those of Myc/Max. WS5 is also related to human glycoprotein GPNMB expressed in metastatic melanoma cells and implicated in the progression of brain and liver tumors.

Using subtractive hybridization techniques, we have isolated a gene termed JAC that is strongly and specifically activated in avian fibroblasts transformed by the v-jun oncogene of avian sarcoma virus 17 (ASV17), but not in cells transformed by other oncogenic agents. Furthermore, JAC is highly expressed in cell lines derived from jun-induced avian fibrosarcomas. Kinetic analysis using a doxycycline-controlled conditional cell transformation system showed that expression of the 0.8-kb JAC mRNA is induced rapidly upon activation of the oncogenic v-jun allele. Nucleotide sequence analysis and transcriptional mapping revealed that the JAC gene contains two exons, with the longest ORF confined to exon 2. The deduced 68-amino acid chicken JAC protein is rich in cysteine residues and displays 37% sequence identity to mammalian highsulfur keratin-associated proteins. The promoter region of JAC contains a consensus (5-TGACTCA-3) and a nonconsensus (5-TGAGTAA-3) AP-1 binding site in tandem, which are both specifically bound by the Gag-Jun hybrid protein encoded by ASV17. Mutational analysis revealed that the two AP-1 sites confer strong transcriptional activation by Gag-Jun in a synergistic manner. Ectopic expression of JAC in avian fibroblasts leads to anchorageindependent growth, strongly suggesting that deregulation of JAC is an essential event in jun-induced cell transformation and tumorigenesis.jun oncogene ͉ signal transduction ͉ gene expression ͉ protein-DNA interaction ͉ transcriptional control

Long-term stability of monoclonal antibodies to be used as biologics is a key aspect in their development. Therefore, its possible early prediction from accelerated stability studies is of major interest, despite currently being regarded as not sufficiently robust. In this work, using a combination of accelerated stability studies (up to 6 months) and first order degradation kinetic model, we are able to predict the long-term stability (up to 3 years) of multiple monoclonal antibody formulations. More specifically, we can robustly predict the long-term stability behaviour of a protein at the intended storage condition (5 °C), based on up to six months of data obtained for multiple quality attributes from different temperatures, usually from intended (5 °C), accelerated (25 °C) and stress conditions (40 °C). We have performed stability studies and evaluated the stability data of several mAbs including IgG1, IgG2, and fusion proteins, and validated our model by overlaying the 95% prediction interval and experimental stability data from up to 36 months. We demonstrated improved robustness, speed and accuracy of kinetic long-term stability prediction as compared to classical linear extrapolation used today, which justifies long-term stability prediction and shelf-life extrapolation for some biologics such as monoclonal antibodies. This work aims to contribute towards further development and refinement of the regulatory landscape that could steer toward allowing extrapolation for biologics during the developmental phase, clinical phase, and also in marketing authorisation applications, as already established today for small molecules.

Fruher ist gezeigt wordenl), daB C h l o r -a c e t y l c h l o r i d , mit 8 -A m i n o -c r o t o n s a u r e e s t e r in Gegenwart von Pyridin in Reaktion gebracht, am Kohlenstoff des Esters uhter Bildung des C h l o r a c e t y l -8a m in 0 -c r o t o n s S u r e e s t e r s, CHa . C (NHs) : C (GO. CHa Cl) . COO. C I H s , angreift, mrahrend A c e t y l -und B e n z o y l c h l o r i d unter den gleichen Betlingungen j e z w e i i s o m e r e , als a-und p-Verbindungen unterschiedene S t i c k s t o f f d e r i v a t e liefern. D a vermutet wurde, dalj ffir den Eintritt des Siiurerestes am Kohlenstoff oder Stickstoff die Starke der dern angewandten Saurechlorid zugrunde liegenden Saure maagebend ware, so wurden verschiedene Siurechloride ausgewahlt und in analoger Weise mit dern Aminoester oder ihm ahnlicher Substaneen zusarnmengebracht, urn so womiiglich eine Gesetz-maBigkeit eu ermitteln, nach der Angriff am Kohlenstoff oder Stickstoff erfolgt. Storencl wirkte bei den Versuchen leider die leichte Spaltbarkeit der Aminoverbindungen durch saure Agenzien. Von den 3 N i t r o -b e n z o y l c h l o r i d e n , die zunachst auf den Aminoester Eur Einwirkung gelangten, fuhrten die m l u -untl pum-Verbindung zu je 2 isomeren Stickstoffderivaten, wie die Spaltbarkeit zu den entsprechenden Nitro-benzamiden ergab; ihr Verhalten ist also dem tles Benzoylchlorids analog. Dagegen lieB sich bei An wentlung von o-Nitrobenzoylchlorid aus dem Reaktionsgeniisch nur o-Nitrobenzamid isolieren. Ebenso lieferten .die 3 B r o m -b e n z o y l c h l o r i d e keine oormalen Produkte; 0-und ni-Brom-benzoylchloridd gaben n u r die entsprechenden Amkle, p-Brom-benzoylchlorid fiihrte nur zum p-Rrombenzoesalire-anhydrid. Versuche mit A c e t y Is a1 icy1 s a u r e c hl o r id verliefen vollig negativ. D i b r o ms a l i c y l s a u r e c h l o r i d gab, mit Amino-crotonester in Reaktion gebracht, n u r eine Substanz, die sich als ein D i b r o m -s a l i c y l i c 1 erwies. Dieses Salicylitl ist verschieden yon dern von A n s c h i i t z 3 ) beim Erhitzen von Dibrom-salicylsaurecblorid im Vakuum uber seinen Schmelzpunkt erhaltenen und auch von dem Produkt, clas bei cler Einwirkung von Pyridin nuf Dibronil ) Die Versuche mit ,&Amino-crotonoster hat F r i t z Keiter, die mit a) B. 41, 3912 [1909]. Acetyl-und Benzoyl-acetonamin H e l e n e S o e n d e r o p ausgefihrt. A. 346, 328 [1906]. aerichte d. D. Chem. Oesellschaft Jahrg. L. 1) Am. SOC. 28, 104 [1906]. 3, Knoevenagel, B. 81, 763 [1898]. ' ) Vorlinder, A. 294, 322 [1897) ' ) o-Brom-benzoylchlorid (vergl. S c h o t t e n , B. 21,2351 [I8881 und S c h o p f , B. 23, 3436 [1890]) siedete unter I 1 mm Druck bei 116O.mem Chloroform gelost und durch Zugabe ron Alkohol xur Abscheidung gebracht. Ag Br. 0.1940 g Shet.: 0.2146 g COz, 0.0192 g H2O. -0.1391 g Shst.: 0.1886 g C~H202Br2. Ber. C 30.23, H 0.72, Br 57.53. GcE. ~3 0 . 1 7 , B 1.11, 57.70. Dernnach liegt ein D i b r o m -s a l i c y l i d vor. Sein Schmelzpunkt liegt bei 3160. Es ist schwer liislich in Chloroform, Benzol, Toluol, Xy...