Immunofluorescence mapping demonstrates that the NG2 proteoglycan is invariably expressed by the mural cell component of mouse neovascular structures. This pattern is independent of the developmental mechanism responsible for formation of the vasculature (vasculogenesis or angiogenesis). Thus, NG2 is expressed in the embryonic heart by cardiomyocytes, in developing macrovasculature by smooth muscle cells, and in nascent microvessels by vascular pericytes. Due to the scarcity of proven markers for developing pericytes, NG2 is especially useful for identification of this cell type. The utility of NG2 as a pericyte marker is illustrated by two observations. First, pericytes are associated with endothelial tubes at an early point in microvessel development. This early interaction between pericytes and endothelial cells has important implications for the role of pericytes in the development and stabilization of microvascular tubes. Second, the pericyte to endothelial cell ratio in developing capillaries varies from tissue to tissue. Because the extent of pericyte investment is likely to affect the physical properties of the vessel in question, it is important to understand the mechanisms that control this process. Additional insight into these and other aspects of vascular morphogenesis should be possible through use of NG2 as a mural cell marker.

Bax inhibitor-1 (BI-1) is an evolutionarily conserved endoplasmic reticulum (ER) protein that suppresses cell death in both animal and plant cells. We characterized mice in which the bi-1 gene was ablated. Cells from BI-1-deficient mice, including fibroblasts, hepatocytes, and neurons, display selective hypersensitivity to apoptosis induced by ER stress agents (thapsigargin, tunicamycin, brefeldin A), but not to stimulators of mitochondrial or TNF/Fas-death receptor apoptosis pathways. Conversely, BI-1 overexpression protects against apoptosis induced by ER stress. BI-1-mediated protection from apoptosis induced by ER stress correlated with inhibition of Bax activation and translocation to mitochondria, preservation of mitochondrial membrane potential, and suppression of caspase activation. BI-1 overexpression also reduces releasable Ca(2+) from the ER. In vivo, bi-1(-/-) mice exhibit increased sensitivity to tissue damage induced by stimuli that trigger ER stress, including stroke and tunicamycin injection. Thus, BI-1 regulates a cell death pathway important for cytopreservation during ER stress.

The Death Inducing Signaling Complex (DISC) formed by Fas receptor, FADD and caspase-8 is a pivotal trigger of apoptosis1-3. The Fas/FADD DISC represents a receptor platform, which once assembled initiates the induction of programmed cell death. A highly oligomeric network of homotypic protein interactions comprised of the death domains (DD) of Fas and FADD is at the center of DISC formation4, 5. Thus characterising the mechanistic basis for the Fas/FADD interaction is paramount for understanding DISC signaling but has remained enigmatic largely due to a lack of structural data. We have successfully formed and isolated the Fas/FADD DD complex and here we report the 2.7 Å crystal structure. The complex shows a tetrameric arrangement of four FADD DDs bound to four Fas DDs. We show that an opening of the Fas DD exposes the FADD binding site and simultaneously generates a Fas/Fas bridge. The result is a regulatory Fas/FADD complex bridge governed by weak protein:protein interactions revealing a model where the complex functions as a mechanistic switch. This switch prevents accidental DISC assembly, yet allows for highly processive DISC formation and clustering upon a sufficient stimulus. Thus besides depicting a previously unknown mode of death domain interactions, these results further uncover a mechanism for receptor signaling solely by oligomerization and clustering events.

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Abstract. We previously described the isolation of mutants of the yeast P~chia pastoris that are deficient in peroxisome assembly (pas mutants). We describe the characterization of one of these mutants, pas8, and the cloning of the PAS8 gene. The pas8 mutant is deficient for growth, but not for division or segregation of peroxisomes, or for induction of peroxisomal proteins. Two distinct peroxisomal targeting signals, PTS1 and PTS2, have been identified that are sufficient to direct proteins to the peroxisomal matrix. We show that the pas8 mutant is deficient in the import of proteins with the PTS1, but not the PTS2, targeting signal. This is the same import deficiency as that found in cells from patients with the lethal human peroxisomal disorder Zellweger syndrome. Cloning and sequencing of the PAS8 gene reveals that it is a novel member of the tetratricopeptide repeat gene family. Antibodies raised against bacterially expressed PAS8 are used to show that PASS is a peroxisomal, membrane-associated protein. Also, we have found that in vitro translated PAS8 protein is capable of binding the PTSl targeting signal specifically, raising the possibility that PAS8 is a PTS1 receptor.T hE compartmentalization of cellular processes into different subcellular compartments is one of the hallmarks of the eukaryotic cell. One such compartment, the peroxisome, is ubiquitously present in virtually all eukaryotic cells. Specialized versions of peroxisome-like compartments (e.g., glycosomes of trypanosomes and glyoxysomes of plants) exist in certain organisms. The peroxisomes, glycosomes, and glyoxysomes have been termed collectively as microbodies. Peroxisomes are characterized by the presence of at least one peroxide-generating oxidase and the enzyme catalase which degrades hydrogen peroxide. Peroxisomes are bounded by a single membrane and vary in size from 0.1 to 1.0 #m in diameter. Like mitochondria, peroxisomes are thought to arise by budding and division of preexisting organelles. Unlike mitochondria, peroxisomes do not contain DNA and must import all constituent proteins.Both peroxisomal matrix and membrane proteins are synthesized in the cytosol on free polysomes and imported posttranslationally into the organdie (for review see Lazarow and Fujiki, 1985).Although the targeting signals for peroxisomal membrane proteins are unknown, recent work has demonstrated the existence of at least two distinct signals that will direct proteins to the peroxisomal matrix (for review see Subramani, 1992).

No abstract

The nature and even existence of adult pancreatic endocrine stem or progenitor cells is a subject of controversy in the field of beta-cell replacement for diabetes. One place to search for such cells is in the nonendocrine fraction of cells that remain after islet isolation, which consist of a mixture of epithelia and mesenchyme. Culture in G418 resulted in elimination of the mesenchymal cells, leaving a highly purified population of nonendocrine pancreatic epithelial cells (NEPECs). To evaluate their differentiation potential, NEPECs were heritably marked and transplanted under the kidney capsule of immunodeficient mice. When cotransplanted with fetal pancreatic cells, NEPECs were capable of endocrine differentiation. We found no evidence of beta-cell replication or cell fusion that could have explained the appearance of insulin positive cells from a source other than NEPECs. Nonendocrine-to-endocrine differentiation of NEPECs supports the existence of endocrine stem or progenitor cells within the epithelial compartment of the adult human pancreas.

MT1-MMP1 (MMP-14) is a member of a large family of zinc endoproteinases, matrixins or matrix metalloproteinases (MMPs) (1, 2). There are several structural features such as the modular domain structure and the existence of an N-terminal propeptide domain, a zinc-coordinating active site domain, and a C-terminal hemopexin-like domain that are characteristic for most MMPs (1-3). A subfamily of membrane type (MT)-MMPs including MT1-MMP is distinguished by a relatively short transmembrane domain and a cytoplasmic tail, which associate these enzymes with discrete regions of the plasma membrane and the intracellular compartment. MT1-MMP expression has been documented in many tumor cell types and strongly implicated in malignant progression (3, 4). In addition to its ability to directly cleave certain components of the extracellular matrix (5, 6), MT1-MMP initiates the activation pathway of the most widespread MMP, MMP-2, by converting pro-MMP-2 into an activation intermediate that further undergoes autocatalytic conversion to generate the mature enzyme of MMP-2 (7-9). Structure-function relationships of MT1-MMP (10 -16) and the mechanisms of pro-MMP-2 activation to the mature enzyme (9,(17)(18)(19)(20) are not understood in detail (21)(22)(23)). An immediate proximity of at least two molecules of MT1-MMP (an "activator" and a "receptor") on the cell surface is required for in trans activation of MMP-2 to the mature form (17,19,20,24). However, there is no direct biochemical evidence to support the existence of MT1-MMP oligomers on cell surfaces. In addition, mechanisms involved in activation and trafficking of MT1-MMP are not well elucidated and remain controversial (10,(13)(14)(15)(25)(26)(27)(28). Thus, furin, a serine proteinase of the trans-Golgi network, has been earlier assumed to function as a unique activator of MT1-MMP (25). However, evidence is emerging that there could be alternative pathways of 28). In this respect, it is not possible to rule out certain autocatalytic steps in MT1-MMP activation such as those involved in the activation pathway of pro-MMP-2 and pro- [29][30][31].To better understand functions of MT1-MMP, we con-* This work was supported by National Institutes of Health Grants CA83017 and CA77470, California Breast Cancer Program Grant 5JB0094, and Susan G. Komen Breast Cancer Foundation Grant 9849 (to A. Y. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.¶ To whom correspondence and reprint requests should be addressed: The Burnham Institute, 10901 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-713-6271; Fax: 858-646-3192; E-mail: strongin@ burnham.org 1 The abbreviations used are: MT, membrane type; MMP, matrix metalloproteinase; BSA, bovine serum albumin; PBS, phosphate-buffered saline; DPBS, Dulbecco's phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; TIMP, tissue inhibitor of me...