ABSTRACT. The Golgi stress response is a mechanism by which, under conditions of insufficient Golgi function (Golgi stress), the transcription of Golgi-related genes is upregulated through an enhancer, the Golgi apparatus stress response element (GASE), in order to maintain homeostasis in the Golgi. The molecular mechanisms associated with GASE remain to be clarified. Here, we identified TFE3 as a GASE-binding transcription factor. TFE3 was phosphorylated and retained in the cytoplasm in normal growth conditions, whereas it was dephosphorylated, translocated to the nucleus and activated Golgi-related genes through GASE under conditions of Golgi stress, e.g. in response to inhibition of oligosaccharide processing in the Golgi apparatus. From these observations, we concluded that the TFE3-GASE pathway is one of the regulatory pathways of the mammalian Golgi stress response, which regulates the expression of glycosylation-related proteins in response to insufficiency of glycosylation in the Golgi apparatus.

Agarose gel electrophoresis of DNA and RNA is routinely performed using buffers containing either Tris, acetate and EDTA (TAE) or Tris, borate and EDTA (TBE). Gels are run at a low, constant voltage (~ 10 V/cm) to minimize current and asymmetric heating effects, which can induce band artifacts and poor resolution. In this study, alterations of gel structure and conductive media composition were analyzed to identify factors causing higher electrical currents during horizontal slab gel electrophoresis. Current was reduced when thinner gels and smaller chamber buffer volumes were used, but was not influenced by agarose concentration or the presence of ethidium bromide. Current was strongly dependent upon the amount and type of EDTA used and on the concentrations of the major acid-base components of each buffer. Interestingly, resolution and the mobilities of circular versus linear plasmid DNAs were also affected by the chemical form and amount of EDTA. With appropriate modifications to gel structure and buffer constituents, electrophoresis could be performed at high voltages (20–25 V/cm), reducing run times by up to 3-fold. The most striking improvements were observed with small DNAs and RNAs (10 – 100 bp): high voltages and short run times produced sharper bands and higher resolution.

Aurora A mitotic kinase is frequently overexpressed in various human cancers and is widely considered to be an oncoprotein. However, the cellular contexts in which Aurora A induces malignancy in vivo are still unclear. We previously reported a mouse model in which overexpression of human Aurora A in the mammary gland leads to small hyperplastic changes but not malignancy because of the induction of p53-dependent apoptosis. To study the additional factors required for Aurora A-associated tumorigenesis, we generated a new Aurora A overexpression mouse model that lacks p53. We present evidence here that Aurora A overexpression in primary mouse embryonic fibroblasts (MEFs) that lack p53 overrides postmitotic checkpoint and leads to the formation of multinucleated polyploid cells. Induction of Aurora A overexpression in the mammary glands of p53-deficient mice resulted in development of precancerous lesions that were histologically similar to atypical ductal hyperplasia in human mammary tissue and showed increased cellular senescence and p16 expression. We further observed DNA damage in p53-deficient primary MEFs after Aurora A overexpression. Our results suggest that Aurora A overexpression in mammary glands is insufficient for the development of malignant tumors in p53-deficient mice because of the induction of cellular senescence. Both p53 and p16 are critical in preventing mammary gland tumorigenesis in the Aurora A overexpression mouse model.

Pressure‐sensitive paint (PSP), which consists of luminescent molecules embedded in an oxygen‐permeable polymer, has been developed for use in wind‐tunnel experiments. To improve the PSP technique, a novel luminescent methacrylate monomer, 5‐[4‐(2‐methacryloyloxyethoxycarbonyl)phenyl]‐10,15,20‐triphenylporphinato platinum(II), was synthesized and copolymerized with isobutyl methacrylate and 2,2,2‐trifluoroethyl methacrylate to produce a dye‐pendant copolymer (2). The introduction of 5,10,15,20‐tetraphenylporphinato platinum(II) (PtTPP) dye into 2 was confirmed by ultraviolet–visible spectroscopy and extended X‐ray absorption fine structure measurements. The extent of PtTPP dye incorporation in 2 was proportional to the molar fraction of the PtTPP‐pendant methacrylate monomer in the feed. The oxygen‐sensing property of 2 was compared with that of a PSP consisting of PtTPP dye embedded in poly(isobutyl‐co‐2,2,2‐trifluoroethyl methacrylate). Although the simple mixture of PtTPP and poly(isobutyl‐co‐2,2,2‐trifluoroethyl methacrylate) showed a marked deviation from a single Stern–Volmer relation, novel copolymer 2 gave a highly linear Stern–Volmer plot. This was unequivocal evidence of dye conjugation on the oxygen‐sensing polymer film. © 2005Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 2997–3006, 2005

Cultural heritage contains a large number of precious proteinaceous specimens, such as wool and silk textiles, leather objects, paper, paint, coatings, binders (and associated adhesives), etc. To minimize the degradation of and to preserve these artifacts, it is desirable to understand the fundamental factors that cause their degradation, to identify the deterioration markers that determine their degradation stage and their age, and to use technologies that can provide this information rapidly while consuming a minimal amount of sample. There are several forces that cause protein degradation, including amino acid racemization, protein deamidation, and protein truncation. The purpose of this paper is to study protein deamidation using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for high-throughput dating of museums wool specimens. For proof of concept, several well-dated sheep's wool textiles from museum collections were analyzed. For wool samples aged from the present to ~400 years ago, the deamidation of two asparagine-containing peptides obtained from the tryptic digest of sheep wool were found to behave linearly in time, indicating that they could act as a potential biomarker of aging for wool samples.