We examined the biosynthesis and post-translational processing of the brain-derived neurotrophic factor precursor (pro-BDNF) in cells infected with a pro-BDNFencoding vaccinia virus. Metabolic labeling, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis reveal that pro-BDNF is generated as a 32-kDa precursor that is N-glycosylated and glycosulfated on a site, within the pro-domain. Some pro-BDNF is released extracellularly and is biologically active as demonstrated by its ability to mediate TrkB phosphorylation. The precursor undergoes N-terminal cleavage within the trans-Golgi network and/or immature secretory vesicles to generate mature BDNF (14 kDa). Small amounts of a 28-kDa protein that is immunoprecipitated with BDNF antibodies is also evident. This protein is generated in the endoplasmic reticulum through N-terminal cleavage of pro-BDNF at the Arg-Gly-Leu-Thr 57 -2-SerLeu site. Cleavage is abolished when Arg 54 is changed to Ala (R54A) by in vitro mutagenesis. Blocking generation of 28-kDa BDNF has no effect on the level of mature BDNF and blocking generation of mature BDNF with ␣ 1 -PDX, an inhibitor of furin-like enzymes, does not lead to accumulation of the 28-kDa form. These data suggest that 28-kDa pro-BDNF is not an obligatory intermediate in the formation of the 14-kDa form in the constitutive secretory pathway. Brain-derived neurotrophic factor (BDNF)1 along with nerve growth factor (NGF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) are members of the neurotrophin family of trophic factors (1). The neurotrophins play essential roles in the development, survival, and function of a wide range of neurons in both the peripheral and central nervous systems.The neurotrophins have a number of shared characteristics, including similar molecular weights (13.2-15.9 kDa), isoelectric points (in the range of 9 -10), and ϳ50% identity in primary structure. They exist in solution as noncovalently bound dimers. Six cysteine residues conserved in the same relative positions give rise to three intra-chain disulfide bonds (2, 3). The neurotrophins interact with two cell surface receptors, the low affinity P75 receptor (4) and the Trk family of high affinity tyrosine kinase receptors (5). NGF preferentially binds TrkA, BDNF and NT4/5 bind TrkB, and NT-3 binds TrkC (and TrkA to a lesser extent).Sequence data predict that mature neurotrophins are generated through the proteolytic processing of higher molecular weight precursors (31-35 kDa), a process that has been extensively studied with respect to the production of NGF (6, 7). Almost nothing is known, however, about the biosynthesis and post-translational processing of the other members of the neurotrophin family. Recent data from our laboratory show that cells with a regulated secretory pathway, including central nervous system neurons, release mature (i.e. fully processed) NGF (8) and NT-3 (9) via the constitutive secretory pathway, whereas mature BDNF is packaged in vesicles and released through the regulated pathway (8). Furthermore, BDNF i...

OCT4 is a master regulator of self-renewal in embryonic stem cells and can potentially encode two spliced variants, designated OCT4A and OCT4B. We have examined the expression pattern of these OCT4 isoforms in various human pluripotent and nonpluripotent cells. Our data revealed that whereas OCT4A expression is restricted to embryonic stem (ES) and embryonal carcinoma (EC) cells, OCT4B can be detected in various nonpluripotent cell types. Furthermore, we detected a novel OCT4 spliced variant, designated OCT4B1, that is expressed primarily in human ES and EC cells and is downregulated following their differentiation. We also found a significantly higher level of OCT4B1 expression in stage-specific embryonic antigen-3 (SSEA3)(؉) compared with SSEA3(؊) subpopulations of cultured ES cells. Taken together, our data demonstrated a distinctive expression pattern for OCT4 spliced variants in different cell types and highlight the necessity of defining the type of OCT4 when addressing the expression of this gene in different human cells. STEM CELLS 2008; 26:3068 -3074 Disclosure of potential conflicts of interest is found at the end of this article.

Using reverse transcriptase-PCR and degenerate oligonucleotides derived from the active-site residues of subtilisin͞kexin-like serine proteinases, we have identified a highly conserved and phylogenetically ancestral human, rat, and mouse type I membrane-bound proteinase called subtilisin͞kexin-isozyme-1 (SKI-1). Computer databank searches reveal that human SKI-1 was cloned previously but with no identified function. In situ hybridization demonstrates that SKI-1 mRNA is present in most tissues and cells. Cleavage specificity studies show that SKI-1 generates a 28-kDa product from the 32-kDa brain-derived neurotrophic factor precursor, cleaving at an RGLT2SL bond. In the endoplasmic reticulum of either LoVo or HK293 cells, proSKI-1 is processed into two membrane-bound forms of SKI-1 (120 and 106 kDa) differing by the nature of their N-glycosylation. Late along the secretory pathway some of the membrane-bound enzyme is shed into the medium as a 98-kDa form. Immunocytochemical analysis of stably transfected HK293 cells shows that SKI-1 is present in the Golgi apparatus and within small punctate structures reminiscent of endosomes. In vitro studies suggest that SKI-1 is a Ca 2؉ -dependent serine proteinase exhibiting a wide pH optimum for cleavage of pro-brainderived neurotrophic factor.

Overall, our findings suggest that exosomal transfer of miR-100 may be a novel mechanism underlying the paracrine effects of MSC-derived exosomes and may provide a means by which these vesicles can modulate vascular responses within the microenvironment of breast cancer cells.