Neural stem cells in the lateral ventricles of the adult mouse CNS participate in repopulation of forebrain structures in vivo and are amenable to in vitro expansion by epidermal growth factor (EGF). There have been no reports of stem cells in more caudal brain regions or in the spinal cord of adult mammals. In this study we found that although ineffective alone, EGF and basic fibroblast growth factor (bFGF) cooperated to induce the proliferation, self-renewal, and expansion of neural stem cells isolated from the adult mouse thoracic spinal cord. The proliferating stem cells, in both primary culture and secondary expanded clones, formed spheres of undifferentiated cells that were induced to differentiate into neurons, astrocytes, and oligodendrocytes. Neural stem cells, whose proliferation was dependent on EGF+bFGF, were also isolated from the lumbar/sacral segment of the spinal cord as well as the third and fourth ventricles (but not adjacent brain parenchyma). Although all of the stem cells examined were similarly multipotent and expandable, quantitative analyses demonstrated that the lateral ventricles (EGF-dependent) and lumbar/sacral spinal cord (EGF+bFGF-dependent) yielded the greatest number of these cells. Thus, the spinal cord and the entire ventricular neuroaxis of the adult mammalian CNS contain multipotent stem cells, present at variable frequency and with unique in vitro activation requirements.

Endoplasmic reticulum oxidoreductases (Eros) are essential for the formation of disulfide bonds. Understanding disulfide bond catalysis in mammals is important because of the involvement of protein misfolding in conditions such as diabetes, arthritis, cancer, and aging. Mammals express two related Ero proteins, Ero1␣ and Ero1␤. Ero1␤ is incompletely characterized but is of physiological interest because it is induced by the unfolded protein response. Here, we show that Ero1␤ can form homodimers and mixed heterodimers with Ero1␣, in addition to Ero-PDI dimers. Ero-Ero dimers require the Ero active site, occur in vivo, and can be modeled onto the Ero1p crystal structure. Our data indicate that the Ero1␤ protein is constitutively strongly expressed in the stomach and the pancreas, but in a cell-specific fashion. In the stomach, selective expression of Ero1␤ occurs in the enzyme-producing chief cells. In pancreatic islets, Ero1␤ expression is high, but is inversely correlated with PDI and PDIp levels, demonstrating that cell-specific differences exist in the regulation of oxidative protein folding in vivo.Protein folding in the ER 5 attracts considerable interest because the failure of a protein to fold can lead to a host of genetic and acquired diseases (1), ranging from cystic fibrosis to ␣1 anti-trypsin deficiency (2). Professional secretory cells in particular must regulate the synthesis of their ER membranes and chaperones to cope with the demands of increased protein production. This is achieved through ER to nucleus signaling pathways, mediated by the trans-membrane associated proteins Ire1␣, PERK, and ATF6 (3). ATF6 and Ire1␣ induce the transcription of XBP1 and the splicing of its mRNA, culminating in the expression of UPR target genes (4). XBP1 is required for B cell maturation into antibody-producing plasma cells (5), and recently, XBP1 and chronic unfolded protein responses have been implicated in obesity and the onset of type 2 diabetes (6), suggesting that targeting physiological unfolded protein responses may have therapeutic value in this disease.Disulfide bond formation is an essential component of the protein folding process, and disulfide bonds are required for structural stability, enzymatic function, and regulation of protein activity (7). The catalytic events involving the oxidation, reduction, and isomerization of disulfide bonds take place in the ER. During protein oxidation, PDI introduces native disulfide bonds into substrate proteins, and is reoxidized by the Ero proteins (Ero1p in yeast, Ero1␣ and Ero1␤ in humans) (8 -11). In yeast, Pdi1p is capable of both oxidizing and isomerizing disulfide bonds, although the relative importance of each function has been debated (12). In humans, PDI also contributes to collagen biosynthesis as a component of the prolyl-4-hydroxlase complex (13) and can act as a component of the ER degradation machinery, particularly with respect to the unfolding and retro-translocation of toxins (14). Numerous PDI homologues exist in yeast (Mpd1p, Mpd2p, Eps1p, and Eug...

Although ocular transport and delivery have been well studied, metabolism in the eye is not well documented, even for clinically available medications such as levobunolol, a potent and nonselective b-adrenergic receptor antagonist. Recently, we reported an in vitro methodology that could be used to evaluate ocular metabolism across preclinical species and humans. The current investigation provides detailed in vitro ocular and liver metabolism of levobunolol in rat, rabbit, and human S9 fractions, including the formation of equipotent active metabolite, dihydrolevobunolol, with the help of high-resolution mass spectrometry. 11 of the 16 metabolites of levobunolol identified herein, including a direct acetyl conjugate of levobunolol observed in all ocular and liver fractions, have not been reported in the literature. The study documents the identification of six human ocular metabolites that have never been reported. The current investigation presents evidence for ocular and hepatic metabolism of levobunolol via non-cytochrome P450 pathways, which have not been comprehensively investigated to date. Our results indicated that rat liver S9 and human ocular S9 fractions formed the most metabolites. Furthermore, liver was a poor in vitro surrogate for eye, and rat and rabbit were poor surrogates for human in terms of the rate and extent of levobunolol metabolism.

1. Topical glaucoma treatments have often been limited by poor absorption and bioavailability. Betaxolol, a selective β1-blocker, has been well studied for its pharmacokinetics and disposition. Limited ocular, betaxolol metabolism data is available despite a growing number of novel ocular treatments. 2. In vitro ocular fractions indicated the formation of an active metabolite, across rat, rabbit and human, which was only observed historically in the liver. 3. Ocular metabolic profiles of preclinical toxicology species, rat and rabbit, were not predictive of human in vitro ocular data. M1 was specific to human and only captured by the liver data. 4. Liver S9 over predicted the extent of ocular metabolism compared to ocular fractions. Rabbit liver S9 fractions demonstrated extensive glucuronidation and higher parent turn-over in 1 h as compared to other matrices. 5. This research assesses in vitro species and organ differences across preclinical species and human. The complex data set highlights the need for an in vitro ocular system to explore poorly documented ocular metabolism.

Metabolism in the eye for any species, laboratory animals or human, is gaining rapid interest as pharmaceutical scientists aim to treat a wide range of so-called incurable ocular diseases. Over a period of decades, reports of metabolic activity toward various drugs and biochemical markers have emerged in select ocular tissues of animals and humans. Ocular cytochrome P450 (P450) enzymes and transporters have been recently reviewed. However, there is a dearth of collated information on non-P450 drug metabolizing enzymes in eyes of various preclinical species and humans in health and disease. In an effort to complement ocular P450s and transporters, which have been well reviewed in the literature, this review is aimed at presenting collective information on non-P450 oxidative, hydrolytic, and conjugative ocular drug metabolizing enzymes. Herein, we also present a list of xenobiotics or drugs that have been reported to be metabolized in the eye.