Heightened expression of tumor necrosis factor alpha, interleukin 1 alpha, and glial fibrillary acidic protein in experimental Creutzfeldt-Jakob disease in mice.

The ultrastructural pathology of myelinated axons in mice infected experimentally with the Fujisaki strain of Creutzfeldt-Jakob disease (CJD) virus is characterized by myelin sheath vacuolation that closely resembles that induced in murine spinal cord organotypic cultures by tumor necrosis factor alpha (TNF-alpha), a cytokine produced by astrocytes and macrophages. To clarify the role of TNF-alpha in experimental CJD, we investigated the expression of TNF-alpha in brain tissues from CJD virus-infected mice at weekly intervals after inoculation by reverse transcription-coupled PCR, Northern and Western blot analyses, and immunocytochemical staining. Neuropathological findings by electron microscopy, as well as expression of interleukin 1 alpha and glial fibrillary acidic protein, were concurrently monitored. As determined by reverse transcription-coupled PCR, the expression of TNF-alpha, interleukin 1 alpha, and glial fibrillary acidic protein was increased by approximately 200-fold in the brains of CJD virus-inoculated mice during the course of disease. By contrast, beta-actin expression remained unchanged. Progressively increased expression of TNF-alpha in CJD virus-infected brain tissues was verified by Northern and Western blot analyses, and astrocytes in areas with striking myelin sheath vacuolation were intensely stained with an antibody against murine TNF-alpha. The collective findings of TNF-alpha overexpression during the course of clinical disease suggest that TNF-alpha may mediate the myelin sheath vacuolation observed in experimental CJD.

Involvement of integrins alpha v beta 3 and alpha v beta 5 in ocular neovascular diseases.

Angiogenesis underlies the majority of eye diseases that result in catastrophic loss of vision. Recent evidence has implicated the integrins alpha v beta 3 and alpha v beta 5 in the angiogenic process. We examined the expression of alpha v beta 3 and alpha v beta 5 in neovascular ocular tissue from patients with subretinal neovascularization from age-related macular degeneration or the presumed ocular histoplasmosis syndrome or retinal neovascularization from proliferative diabetic retinopathy (PDR). Only alpha v beta 3 was observed on blood vessels in ocular tissues with active neovascularization from patients with age-related macular degeneration or presumed ocular histoplasmosis, whereas both alpha v beta 3 and alpha v beta 5 were present on vascular cells in tissues from patients with PDR. Since we observed both integrins on vascular cells from tissues of patients with retinal neovascularization from PDR, we examined the effects of a systemically administered cyclic peptide antagonist of alpha v beta 3 and alpha v beta 5 on retinal angiogenesis in a murine model. This antagonist specifically blocked new blood vessel formation with no effect on established vessels. These results not only reinforce the concept that retinal and subretinal neovascular diseases are distinct pathological processes, but that antagonists of alpha v beta 3 and/or alpha v beta 5 may be effective in treating individuals with blinding eye disease associated with angiogenesis.

The differential adhesion forces of anterior cruciate and medial collateral ligament fibroblasts: effects of tropomodulin, talin, vinculin, and alpha-actinin.

We have determined the effects of tropomodulin (Tmod), talin, vinculin, and alpha-actinin on ligament fibroblast adhesion. The anterior cruciate ligament (ACL), which lacks a functional healing response, and the medial collateral ligament (MCL), a functionally healing ligament, were selected for this study. The micropipette aspiration technique was used to determine the forces needed to separate ACL and MCL cells from a fibronectin-coated surface. Delivery of exogenous tropomodulin, an actin-filament capping protein, into MCL fibroblasts significantly increased adhesion, whereas its monoclonal antibody (mAb) significantly decreased cell adhesiveness. However, for ACL fibroblasts, Tmod significantly reduced adhesion, whereas its mAb had no effect. mAbs to talin, vinculin, and alpha-actinin significantly decreased the adhesion of both ACL and MCL cells with increasing concentrations of antibody, and also reduced stress fiber formation and cell spreading rate as revealed by immunofluorescence microscopy. Disruption of actin filament and microtubule assembly with cytochalasin D and colchicine, respectively, also significantly reduced adhesion in ACL and MCL cells. In conclusion, both ACL and MCL fibroblast adhesion depends on cytoskeletal assembly; however, this dependence differs between ACL and MCL fibroblasts in many ways, especially in the role of Tmod. These results add yet another possible factor in explaining the clinical differences in healing between the ACL and the MCL.

Cleavage of lamin A by Mch2 alpha but not CPP32: multiple interleukin 1 beta-converting enzyme-related proteases with distinct substrate recognition properties are active in apoptosis.

Although proteases related to the interleukin 1 beta-converting enzyme (ICE) are known to be essential for apoptotic execution, the number of enzymes involved, their substrate specificities, and their specific roles in the characteristic biochemical and morphological changes of apoptosis are currently unknown. These questions were addressed using cloned recombinant ICE-related proteases (IRPs) and a cell-free model system for apoptosis (S/M extracts). First, we compared the substrate specificities of two recombinant human IRPs, CPP32 and Mch2 alpha. Both enzymes cleaved poly-(ADP-ribose) polymerase, albeit with different efficiencies. Mch2 alpha also cleaved recombinant and nuclear lamin A at a conserved VEID decreases NG sequence located in the middle of the coiled-coil rod domain, producing a fragment that was indistinguishable from the lamin A fragment observed in S/M extracts and in apoptotic cells. In contrast, CPP32 did not cleave lamin A. The cleavage of lamin A by Mch2 alpha and by S/M extracts was inhibited by millimolar concentrations of Zn2+, which had a minimal effect on cleavage of poly (ADP-ribose) polymerase by CPP32 and by S/M extracts. We also found that N-(acetyltyrosinylvalinyl-N epsilon-biotinyllysyl)aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone, which derivatizes the larger subunit of active ICE, can affinity label up to five active IRPs in S/M extracts. Together, these observations indicate that the processing of nuclear proteins in apoptosis involves multiple IRPs having distinct preferences for their apoptosis-associated substrates.

16 alpha-substituted analogs of the antiprogestin RU486 induce a unique conformation in the human progesterone receptor resulting in mixed agonist activity.

Previously, we have shown that agonists and antagonists interact with distinct, though overlapping regions within the human progesterone receptor (hPR) resulting in the formation of structurally different complexes. Thus, a link was established between the structure of a ligand-receptor complex and biological activity. In this study, we have utilized a series of in vitro assays with which to study hPR pharmacology and have identified a third class of hPR ligands that induce a receptor conformation which is distinct from that induced by agonists or antagonists. Importantly, when assayed on PR-responsive target genes these compounds were shown to exhibit partial agonist activity; an activity that was influenced by cell context. Thus, as has been shown previously for estrogen receptor, the overall structure of the ligand-receptor complex is influenced by the nature of the ligand. It appears, therefore, that the observed differences in the activity of some PR and estrogen receptor ligands reflect the ability of the cellular transcription machinery to discriminate between the structurally different complexes that result following ligand interaction. These data support the increasingly favored hypothesis that different ligands can interact with different regions within the hormone binding domains of steroid hormone receptors resulting in different biologies.

Crystal structure of D-amino acid oxidase: a case of active site mirror-image convergent evolution with flavocytochrome b2.

D-amino acid oxidase is the prototype of the FAD-dependent oxidases. It catalyses the oxidation of D-amino acids to the corresponding alpha-ketoacids. The reducing equivalents are transferred to molecular oxygen with production of hydrogen peroxide. We have solved the crystal structure of the complex of D-amino acid oxidase with benzoate, a competitive inhibitor of the substrate, by single isomorphous replacement and eightfold averaging. Each monomer is formed by two domains with an overall topology similar to that of p-hydroxybenzoate hydroxylase. The benzoate molecule lays parallel to the flavin ring and is held in position by a salt bridge with Arg-283. Analysis of the active site shows that no side chains are properly positioned to act as the postulated base required for the catalytic carboanion mechanism. On the contrary, the benzoate binding mode suggests a direct transfer of the substrate alpha-hydrogen to the flavin during the enzyme reductive half-reaction.The active site Of D-amino acid oxidase exhibits a striking similarity with that of flavocytochrome b2, a structurally unrelated FMN-dependent flavoenzyme. The active site groups (if these two enzymes are in fact superimposable once the mirror-image of the flavocytochrome b2 active site is generated with respect to the flavin plane. Therefore, the catalytic sites of D-amino acid oxidase and flavocytochrome b2 appear to have converged to a highly similar but enantiomeric architecture in order to catalvze similar reactions (oxidation of alpha-amino acids or alpha-hydroxy acids), although with opposite stereochemistry.

Complete structure of the human alpha-albumin gene, a new member of the serum albumin multigene family.

The nucleotide sequence of the human alpha-albumin gene, including 887 bp of the 5'-flanking region and 1311 bp of the 3-flanking region (24,454 in total), was determined from three overlapping lambda phage clones. The sequence spans 22,256 bp from the cap site to the polyadenylylation site, revealing a gene structure of 15 exons separated by 14 introns. The methionine initiation codon ATG is within exon 1; the termination codon TGA is within exon 14. Exon 15 is entirely untranslated and contains the polyadenylylation signal AATAAA. The deduced polypeptide chain is composed of a 21-amino-acid leader peptide, followed by 578 amino acids of the mature protein. There are seven repetitive DNA elements (Alu and Kpn) in the introns and 3-flanking region. The sizes of the 15 alpha-albumin exons match closely those of the albumin, alpha-fetoprotein, and vitamin D-binding protein genes. The exons are symmetrically placed within the three domains of the individual proteins, and they share a characteristic codon splitting pattern that is conserved among members of the gene family. The results provide strong evidence that alpha-albumin belongs to, and most likely completes with, the serum albumin gene family. Based on structural similarity, alpha-albumin appears to be most closely related to alpha-fetoprotein. The complete structure of this family of four tandemly linked genes provides a well-characterized approximately 200 kb locus in the 4q subcentromeric region of the human genome.

Transcriptionally active Stat1 is required for the antiproliferative effects of both interferon alpha and interferon gamma.

Type I (alpha, beta) and type II (gamma) interferons (IFNs) can restrict the growth of many cell types. INF-stimulated gene transcription, a key early event in IFN response, acts through the Janus kinase-signal transducers and activators of transcription pathway, in which both IFN-alpha and IFN-gamma activate the transcription factor Stat1. A cell line lacking Stat1 (U3A) was not growth-arrested by IFN-alpha or IFN-gamma, and experiments were carried out with U3A cells permanently expressing normal or various mutant forms of Stat1 protein. Only cells in which complete Stat1 activity was available (Stat1alpha) were growth-inhibited by IFN-gamma. A mutant that supports 20-30% normal transcription did not cause growth restraint. In contrast, IFN-alpha growth restraint was imposed by cells producing Stat1beta, which lacks transcriptional activation potential. This parallels earlier results showing the truncated Stat1 can function in IFN-alpha gene activation. In addition to experiments on long-term cultured cells, we also found that wild-type primary mouse embryonic fibroblasts were inhibited by IFNs, but fibroblasts from Stat1-deficient mouse embryos were not inhibited by IFNs.

The glucose sensor protein glucokinase is expressed in glucagon-producing alpha-cells.

Expression of glucokinase in hepatocytes and pancreatic 6-cells is of major physiologic importance to mammalian glucose homeostasis. Liver glucokinase catalyzes the first committed step in the disposal of glucose, and beta-cell glucokinase catalyzes a rate-limiting step required for glucose-regulated insulin release. The present study reports the expression of glucokinase in rat glucagon-producing alpha-cells, which are negatively regulated by glucose. Purified rat alpha-cells express glucokinase mRNA and protein with the same transcript length, nucleotide sequence, and immunoreactivity as the beta-cell isoform. Glucokinase activity accounts for more than 50% of glucose phosphorylation in extracts of alpha-cells and for more than 90% of glucose utilization in intact cells. The glucagon-producing tumor MSL-G-AN also contained glucokinase mRNA, protein, and enzymatic activity. These data indicate that glucokinase may serve as a metabolic glucose sensor in pancreatic alpha-cells and, hence, mediate a mechanism for direct regulation of glucagon release by extracellular glucose. Since these cells do not express Glut2, we suggest that glucose sensing does not necessarily require the coexpression of Glut2 and glucokinase.

Taste receptor-like cells in the rat gut identified by expression of alpha-gustducin.

The alpha-subunit of the trimeric G-protein complex specific for taste receptor cells of the tongue, alpha-gustducin, is described here to be also expressed in the stomach and intestine. The alpha-gustducin-containing cells were identified as brush cells that are scattered throughout the surface epithelium of the gut and share structural features of taste receptor cells of the tongue. These findings provide clues to the long-sought molecular and cellular basis for chemoreception in the gut.

Different functions for the interleukin 8 receptors (IL-8R) of human neutrophil leukocytes: NADPH oxidase and phospholipase D are activated through IL-8R1 but not IL-8R2.

Two monoclonal antibodies, anti-IL8R1 and anti-IL8R2, raised against both interleukin 8 receptors (IL-8R) of human neutrophils, IL-8R1 and IL-8R2, were used to study individual receptor functions after stimulation with IL-8, GRO alpha, or NAP-2. Efficacy and selectivity of the antibodies were tested in Jurkat cells transfected with cDNA coding for one or the other receptor. The binding of 125 I labeled IL-8 and IL-8-induced changes of the cytosolic free Ca2+ concentration were inhibited by anti-IL8RI in cells expressing IL-8R1 and by anti-IL8R2 in cells expressing IL-8R2. In human neutrophils, release of elastase was observed after stimulation with IL-8 or GRO alpha. The response to IL-8 was inhibited slightly by anti-IL8R1 and more substantially when both monoclonal antibodies were present, while the response to GRO alpha was inhibited by anti-IL8R2 but was not affected by anti-IL8R1. These results indicate that both IL-8 receptors can signal independently for granule enzyme release. Superoxide production, a measure of the respiratory burst, was obtained with increasing concentrations of IL-8 with maximum effects at 25 to 50 nM, but no response was observed upon challenge with GRO alpha or NAP-2 up to 1000 nM. The superoxide production induced by IL-8 was inhibited by anti-IL8R1, but was not affected by anti-IL8R2. Stimulation of neutrophils with IL-8, in contrast to GRO alpha or NAP-2, also elicited phospholipase D activity. The effect of IL-8 was again inhibited by anti-IL-8R1 but not by anti-IL8R2, indicating that this response, like the respiratory burst, was mediated by IL-8R1. Taken together, our results show that IL-8R1 and IL-8R2 are functionally different. Responses, such as cytosolic free Ca2+ changes and the release of granule enzymes, are mediated through both receptors, whereas the respiratory burst and the activation of phospholipase D depend exclusively on stimulation through IL-8R1.

A new endogenous natriuretic factor: LLU-alpha.

For over three decades, renal physiology has sought a putative natriuretic hormone (third factor) that might control the body's pool of extracellular fluid, an important determinant in hypertension, congestive heart failure, and cirrhosis. In our search for this hormone, we have isolated several pure natriuretic factors from human uremic urine that would appear, alone or in combination, to mark a cluster of phenomena previously presumed to be that of a single "natriuretic hormone." This paper reports the purification, chemical structure, and total synthesis of the first of these compounds, LLU-alpha, which proved to be 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman, presumably a metabolite of gamma-tocopherol. Both natural LLU-alpha and synthetic material are identical (except for optical activity) with respect to structure and biological activity. It appears that the natriuretic activity of LLU-alpha is mediated by inhibition of the 70 pS K+ channel in the apical membrane of the thick ascending limb of the kidney.

Predominant role of alpha 4-integrins for distinct steps of lymphoma metastasis.

To analyze the role of alpha4-integrins in lymphoma metastasis, sublines of the T-cell lymphoma LB were generated by retrovirus-mediated gene transfer that differ exclusively in the expression of alpha4-integrins. Using LB-alpha4 and control LB-NTK cells, we demonstrate that expression of alpha4-integrins strongly suppresses metastasis formation of LB lymphoma cells in secondary lymphoid organs such as spleen, mesenteric and peripheral lymph nodes, or Peyer's patches after i.v. injection into syngeneic BALB/c mice. Moreover, alpha4-integrin expression inhibited development of metastatic tumors in liver, lung, and kidney. Expansion of LB lymphoma cells in bone marrow was not affected by alpha4-integrin expression. In vivo migration assays using 51Cr-labeled lymphoma cells demonstrated that low-metastatic LB-alpha4 cells accumulated with the same efficiency as high-metastatic LB-NTK cells in all target organs examined and were even enriched in mucosal lymphoid organs. Collectively, these results indicate that alpha4-integrins inhibit metastasis formation of lymphoma cells at a stage subsequent to the invasion of target organs.

Group B streptococci escape host immunity by deletion of tandem repeat elements of the alpha C protein.

Group B streptococci (GBS) are the most common cause of neonatal sepsis, pneumonia, and meningitis. The alpha C protein is a surface-associated antigen; the gene (bca) for this protein contains a series of tandem repeats (each encoding 82 aa) that are identical at the nucleotide level and express a protective epitope. We previously reported that GBS isolates from two of 14 human maternal and neonatal pairs differed in the number of repeats contained in their alpha C protein; in both pairs, the alpha C protein of the neonatal isolate was smaller in molecular size. We now demonstrate by PCR that the neonatal isolates contain fewer tandem repeats. Maternal isolates were susceptible to opsonophagocytic killing in the presence of alpha C protein-specific antiserum, whereas the discrepant neonatal isolates proliferated. An animal model was developed to further study this phenomenon. Adult mice passively immunized with antiserum to the alpha C protein were challenged with an alpha C protein-expressing strain of GBS. Splenic isolates of GBS from these mice showed a high frequency of mutation in bca--most commonly a decrease in repeat number. Isolates from non-immune mice were not altered. Spontaneous deletions in the repeat region were observed at a much lower frequency (6 x 10(-4)); thus, deletions in that region are selected for under specific antibody pressure and appear to lower the organism's susceptibility to killing by antibody specific to the alpha C protein. This mechanism of antigenic variation may provide a means whereby GBS evade host immunity.

Mammalian phospholipase D: phosphatidylethanolamine as an essential component.

Bovine kidney phospholipase D (PLD) was assayed by measuring the formation of phosphatidylethanol from added radioactive phosphatidylcholine (PtdCho) in the presence of ethanol, guanosine 5'-[gamma-thio]triphosphate, ammonium sulfate, and cytosol factor that contained small GTP-binding regulatory proteins. The PLD enzyme associated with particulate fractions was solubilized by deoxycholate and partially purified by chromatography on a heparin-Sepharose column. This PLD preferentially used PtdCho as substrate. After purification, the enzyme per se showed little or practically no activity but required an additional factor for the enzymatic reaction. This factor was extracted with chloroform/methanol directly from particulate fractions of various tissues, including kidney, liver, and brain, and identified as phosphatidylethanolamine (PtdEtn), although this phospholipid did not serve as a good substrate. Plasmalogen-rich PtdEtn, dioleoyl-PtdEtn, and L-alpha-palmitoyl-beta-linoleoyl-PtdEtn were effective, but dipalmitoyl-PtdEtn was inert. Sphingomyelin was 30% as active as PtdEtn. The results suggest that mammalian PLD reacts nearly selectively with PtdCho in the form of mixed micelles or membranes with other phospholipids, especially PtdEtn.