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Alpha-dystroglycan (αDG) is a highly glycosylated cell surface protein with a significant role in cell-to-extracellular matrix interactions in muscle. αDG interaction with extracellular ligands relies on the activity of the LARGE1 glycosyltransferase that synthesizes and extends the heteropolysaccharide matriglycan. Abnormalities in αDG glycosylation and formation of matriglycan are the pathogenic mechanisms for the dystroglycanopathies, a group of congenital muscular dystrophies. Muscle biopsies were evaluated from related 6-week-old Labrador retriever puppies with poor suckling, small stature compared to normal litter mates, bow-legged stance and markedly elevated creatine kinase activities. A dystrophic phenotype with marked degeneration and regeneration, multifocal mononuclear cell infiltration and endomysial fibrosis was identified on muscle cryosections. Single nucleotide polymorphism (SNP) array genotyping data on the family members identified three regions of homozygosity in 4 cases relative to 8 controls. Analysis of whole genome sequence data from one of the cases identified a stop codon mutation in the LARGE1 gene that truncates 40% of the protein. Immunofluorescent staining and western blotting demonstrated the absence of matriglycan in skeletal muscle and heart from affected dogs. Compared to control, LARGE enzyme activity was not detected. This is the first report of a dystroglycanopathy in dogs.

Mutations in multiple genes required for proper O-mannosylation of α-dystroglycan are causal for congenital/limb-girdle muscular dystrophies and abnormal brain development in mammals. Previously, we and others further elucidated the functional O-mannose glycan structure that is terminated by matriglycan, [(-GlcA-β3-Xyl-α3-)n]. This repeating disaccharide serves as a receptor for proteins in the extracellular matrix. Here, we demonstrate in vitro that HNK-1 sulfotransferase (HNK-1ST/carbohydrate sulfotransferase) sulfates terminal glucuronyl residues of matriglycan at the 3-hydroxyl and prevents further matriglycan polymerization by the LARGE1 glycosyltransferase. While α-dystroglycan isolated from mouse heart and kidney is susceptible to exoglycosidase digestion of matriglycan, the functional, lower molecular weight α-dystroglycan detected in brain, where HNK-1ST expression is elevated, is resistant. Removal of the sulfate cap by a sulfatase facilitated dual-glycosidase digestion. Our data strongly support a tissue specific mechanism in which HNK-1ST regulates polymer length by competing with LARGE for the 3-position on the nonreducing GlcA of matriglycan.

Significance Influenza A virus (IAV) is a major cause of respiratory infections. We show that mice lacking the N-terminal domain of α-dystroglycan (α-DGN) exhibit significantly higher viral titers in the lungs after IAV infection. In addition, we show that overexpression of α-DGN in the lungs, both prior and during IAV infection, significantly reduces viral load and that recombinant α-DGN disrupts hemagglutination mediated by the influenza virus. Collectively, we uncover a protective role for α-DGN in IAV proliferation, suggesting it may have antiviral properties and could potentially be used as a treatment for IAV infection. As α-DGN levels are altered in more (inflammatory) disease states, this insight opens new avenues of investigation into the role of α-DGN in inflammation., α-Dystroglycan (α-DG) is a highly glycosylated basement membrane receptor that is cleaved by the proprotein convertase furin, which releases its N-terminal domain (α-DGN). Before cleavage, α-DGN interacts with the glycosyltransferase LARGE1 and initiates functional O-glycosylation of the mucin-like domain of α-DG. Notably, α-DGN has been detected in a wide variety of human bodily fluids, but the physiological significance of secreted α-DGN remains unknown. Here, we show that mice lacking α-DGN exhibit significantly higher viral titers in the lungs after Influenza A virus (IAV) infection (strain A/Puerto Rico/8/1934 H1N1), suggesting an inability to control virus load. Consistent with this, overexpression of α-DGN before infection or intranasal treatment with recombinant α-DGN prior and during infection, significantly reduced IAV titers in the lungs of wild-type mice. Hemagglutination inhibition assays using recombinant α-DGN showed in vitro neutralization of IAV. Collectively, our results support a protective role for α-DGN in IAV proliferation.

Matriglycan [-GlcA-β1,3-Xyl-α1,3-]n serves as a scaffold in many tissues for extracellular matrix proteins containing laminin-G domains including laminin, agrin, and perlecan. Like-acetyl-glucosaminyltransferase 1 (LARGE1) synthesizes and extends matriglycan on α-dystroglycan (α-DG) during skeletal muscle differentiation and regeneration; however, the mechanisms which regulate matriglycan elongation are unknown. Here, we show that Protein O-Mannose Kinase (POMK), which phosphorylates mannose of core M3 (GalNAc-β1,3-GlcNAc-β1,4-Man) preceding matriglycan synthesis, is required for LARGE1-mediated generation of full-length matriglycan on α-DG (~150 kDa). In the absence of Pomk gene expression in mouse skeletal muscle, LARGE1 synthesizes a very short matriglycan resulting in a ~ 90 kDa α-DG which binds laminin but cannot prevent eccentric contraction-induced force loss or muscle pathology. Solution NMR spectroscopy studies demonstrate that LARGE1 directly interacts with core M3 and binds preferentially to the phosphorylated form. Collectively, our study demonstrates that phosphorylation of core M3 by POMK enables LARGE1 to elongate matriglycan on α-DG, thereby preventing muscular dystrophy.

Background Caveolin-3 (CAV3) is a muscle-specific protein localized to the sarcolemma. It was suggested that CAV3 is involved in the connection between the extracellular matrix (ECM) and the cytoskeleton. Caveolinopathies often go along with increased CK levels indicative of sarcolemmal damage. So far, more than 40 dominant pathogenic mutations have been described leading to several phenotypes many of which are associated with a mis-localization of the mutant protein to the Golgi. Golgi retention and endoplasmic reticulum (ER) stress has been demonstrated for the CAV3 p.P104L mutation, but further downstream pathophysiological consequences remained elusive so far. Methods We utilized a transgenic (p.P104L mutant) mouse model and performed proteomic profiling along with immunoprecipitation, immunofluorescence and immunoblot examinations (including examination of α-dystroglycan glycosylation), and morphological studies (electron and coherent anti-Stokes Raman scattering (CARS) microscopy) in a systematic investigation of molecular and subcellular events in p.P104L caveolinopathy. Results Our electron and CARS microscopic as well as immunological studies revealed Golgi and ER proliferations along with a build-up of protein aggregates further characterized by immunoprecipitation and subsequent mass spectrometry. Molecular characterization these aggregates showed affection of mitochondrial and cytoskeletal proteins which accords with our ultra-structural findings. Additional global proteomic profiling revealed vulnerability of 120 proteins in diseased quadriceps muscle supporting our previous findings and providing more general insights into the underlying pathophysiology. Moreover, our data suggested that further DGC components are altered by the perturbed protein processing machinery but are not prone to form aggregates whereas other sarcolemmal proteins are ubiquitinated or bind to p62. Although the architecture of the ER and Golgi as organelles of protein glycosylation are altered, the glycosylation of α-dystroglycan presented unchanged. Conclusions Our combined data classify the p.P104 caveolinopathy as an ER-Golgi disorder impairing proper protein processing and leading to aggregate formation pertaining proteins important for mitochondrial function, cytoskeleton, ECM remodeling and sarcolemmal integrity. Glycosylation of sarcolemmal proteins seems to be normal. The new pathophysiological insights might be of relevance for the development of therapeutic strategies for caveolinopathy patients targeting improved protein folding capacity. Electronic supplementary material The online version of this article (10.1186/s13395-018-0173-y) contains supplementary material, which is available to authorized users.

Background α-Dystroglycan is the highly glycosylated component of the dystrophin-glycoprotein complex (DGC) that binds with high-affinity to extracellular matrix (ECM) proteins containing laminin-G-like (LG) domains via a unique heteropolysaccharide [-GlcA-beta1,3-Xyl-alpha1,3-]n called matriglycan. Changes in expression of components of the DGC or in the O-glycosylation of α-dystroglycan result in muscular dystrophy but are also observed in certain cancers. In mice, the loss of either of two DGC proteins, dystrophin or α-sarcoglycan, is associated with a high incidence of rhabdomyosarcoma (RMS). In addition, glycosylation of α-dystroglycan is aberrant in a small cohort of human patients with RMS. Since both the glycosylation of α-dystroglycan and its function as an ECM receptor require over 18 post-translational processing enzymes, we hypothesized that understanding its role in the pathogenesis of RMS requires a complete analysis of the expression of dystroglycan-modifying enzymes and the characterization of α-dystroglycan glycosylation in the context of RMS. Methods A series of cell lines and biopsy samples from human and mouse RMS were analyzed for the glycosylation status of α-dystroglycan and for expression of the genes encoding the responsible enzymes, in particular those required for the addition of matriglycan. Furthermore, the glycosyltransferase LARGE1 was ectopically expressed in RMS cells to determine its effects on matriglycan modifications and the ability of α-dystroglycan to function as a laminin receptor. Results Immunohistochemistry and immunoblotting of a collection of primary RMS tumors show that although α-dystroglycan is consistently expressed and glycosylated in these tumors, α-dystroglycan lacks matriglycan and the ability to bind laminin. Similarly, in a series of cell lines derived from human and mouse RMS, α-dystroglycan lacks matriglycan modification and the ability to bind laminin. RNAseq data from RMS cell lines was analyzed for expression of the genes known to be involved in α-dystroglycan glycosylation, which revealed that, for most cell lines, the lack of matriglycan can be attributed to the downregulation of the dystroglycan-modifying enzyme LARGE1. Ectopic expression of LARGE1 in these cell cultures restored matriglycan to levels comparable to those in muscle and restored high-affinity laminin binding to α-dystroglycan. Conclusions Collectively, our findings demonstrate that a lack of matriglycan on α-dystroglycan is a common feature in RMS due to the downregulation of LARGE1, and that ectopic expression of LARGE1 can restore matriglycan modifications and the ability of α-dystroglycan to function as an ECM receptor.

Dystroglycan (DG) is a highly expressed extracellular matrix receptor that is linked to the cytoskeleton in skeletal muscle. DG is critical for the function of skeletal muscle, and muscle with primary defects in the expression and/or function of DG throughout development has many pathological features and a severe muscular dystrophy phenotype. In addition, reduction in DG at the sarcolemma is a common feature in muscle biopsies from patients with various types of muscular dystrophy. However, the consequence of disrupting DG in mature muscle is not known. Here, we investigated muscles of transgenic mice several months after genetic knockdown of DG at maturity. In our study, an increase in susceptibility to contraction-induced injury was the first pathological feature observed after the levels of DG at the sarcolemma were reduced. The contraction-induced injury was not accompanied by increased necrosis, excitation-contraction uncoupling, or fragility of the sarcolemma. Rather, disruption of the sarcomeric cytoskeleton was evident as reduced passive tension and decreased titin immunostaining. These results reveal a role for DG in maintaining the stability of the sarcomeric cytoskeleton during contraction and provide mechanistic insight into the cause of the reduction in strength that occurs in muscular dystrophy after lengthening contractions.

Mutations in the LARGE gene have been identified in congenital muscular dystrophy (CMD) patients with brain abnormalities. Both LARGE and its paralog, LARGE2 (also referred to as GYLTL1B) are bifunctional glycosyltransferases with xylosyltransferase (Xyl-T) and glucuronyltransferase (GlcA-T) activities, and are capable of forming polymers consisting of [-3Xyl-α1,3GlcAβ1-] repeats. LARGE-dependent modification of α-dystroglycan (α-DG) with these polysaccharides is essential for the ability of α-DG to act as a receptor for ligands in the extracellular matrix. Here we report on the endogenous enzymatic activities of LARGE and LARGE2 in mice and humans, using a newly developed assay for GlcA-T activity. We show that normal mouse and human cultured cells have endogenous LARGE GlcA-T, and that this activity is absent in cells from the Large(myd) (Large-deficient) mouse model of muscular dystrophy, as well as in cells from CMD patients with mutations in the LARGE gene. We also demonstrate that GlcA-T activity is significant in the brain, heart, and skeletal muscle of wild-type and Large2(-/-) mice, but negligible in the corresponding tissues of the Large(myd) mice. Notably, GlcA-T activity is substantial, though reduced, in the kidneys of both the Large(myd) and Large2(-/-) mice, consistent with the observation of α-DG/laminin binding in these contexts. This study is the first to test LARGE activity in samples as small as cryosections and, moreover, provides the first direct evidence that not only LARGE, but also LARGE2, is vital to effective functional modification of α-DG in vivo.

The ‘pseudokinase’ SgK196 is a protein O-mannose kinase (POMK) that catalyzes an essential phosphorylation step during biosynthesis of the laminin-binding glycan on α-dystroglycan. However, the catalytic mechanism underlying this activity remains elusive. Here we present the crystal structure of Danio rerio POMK in complex with Mg2+ ions, ADP, aluminum fluoride, and the GalNAc-β3-GlcNAc-β4-Man trisaccharide substrate, thereby providing a snapshot of the catalytic transition state of this unusual kinase. The active site of POMK is established by residues located in non-canonical positions and is stabilized by a disulfide bridge. GalNAc-β3-GlcNAc-β4-Man is recognized by a surface groove, and the GalNAc-β3-GlcNAc moiety mediates the majority of interactions with POMK. Expression of various POMK mutants in POMK knockout cells further validated the functional requirements of critical residues. Our results provide important insights into the ability of POMK to function specifically as a glycan kinase, and highlight the structural diversity of the human kinome. DOI: http://dx.doi.org/10.7554/eLife.22238.001

Background. To investigate whether human papilloma virus (HPV)-specific immune mechanisms can result in tumor clearance, we have created HPV+ and HPV− tonsil cells that form squamous cancers in immune-competent mice. Here, we determine that an immune-specific response can clear HPV+ tumor cells and the cellular requirements to mediate this tumor clearance. Methods. Through the benefit of this model, we use in vitro and in vivo methods to better understand how HPV+ cells are rejected. Results. The data show that an in vivo antigen-specific antitumor response is generated to HPV+ transformed cells and that this response requires CD4+ and CD8+ cells to mount this antitumor response. Conclusion. The findings from this preclinical model will have implications not only in understanding human disease but also as a valuable model for testing immunotherapeutic strategies for HPV+ head and neck cancer. © 2009 Wiley Periodicals, Inc. Head Neck, 2009

The human papillomavirus (HPV) oncogene E6 has been shown to perform multiple functions (p53 degradation, telomerase activation, etc.) that play a role in oncogenic transformation. Beyond known E6 functions, an undefined mechanism that allows cellular invasion requires the E6 PDZ binding motif (PDZBM). Here, we show that HPV type 16 (HPV16) E6 interacts with and induces loss of a protein tyrosine phosphatase (PTPN13) in a PDZBM-dependent manner. PTPN13 loss induced either by the presence of E6 or by a short hairpin RNA strategy allows for anchorage-independent growth (AIG) and synergy with a known oncogene, Ras v12 , resulting in invasive growth in vivo. Restoring PTPN13 expression reverses AIG in cells lacking PTPN13. A genomic analysis of colorectal carcinoma has identified an association between PTPN13 loss-of-function mutations and aberrant Ras signaling. Our findings support this correlation and provide methods for further evaluation of the mechanisms by which PTPN13 loss/Ras expression leads to invasive growth, the results of which will be important for treatment of HPV-related and non-HPV cancer.

A function of the transcription factor REST is to block the expression of neuronal phenotypic traits in non-neuronal cells. Previous studies have shown that REST-mediated repression requires histone deacetylase activity and that recruitment of deacetylases is mediated by two co-repressors, Sin3A and CoREST. In this study, we show that a repressor domain in CoREST interacts with BRG1-associated factor (BAF) 57, a component of the hSWI.SNF complex. In vivo, BAF57 occupies the neuronal sodium channel gene (Nav1.2) promoter, and targeting to this gene requires REST. In addition to BAF57, the ATPase BRG1 and BAF170, other members of the hSWI.SNF complex, are also present in the REST.CoREST repressor complex. Microinjection of specific antibodies against BRG1, BAF57, or BAF170 into Rat1 fibroblasts relieves repression of RE1 reporter genes. Together, our data suggest that ATP-dependent chromatin remodeling, as well as histone deacetylation, is needed for REST-mediated repression.

The transcriptional repressor, REST, helps restrict neuronal traits to neurons by blocking their expression in nonneuronal cells. To examine the repercussions of REST expression in neurons, we generated a neuronal cell line that expresses REST conditionally. REST expression inhibited differentiation by nerve growth factor, suppressing both sodium current and neurite growth. A novel corepressor complex, CoREST/HDAC2, was shown to be required for REST repression. In the presence of REST, the CoREST/HDAC2 complex occupied the native Nav1.2 sodium channel gene in chromatin. In neuronal cells that lack REST and express sodium channels, the corepressor complex was not present on the gene. Collectively, these studies define a novel HDAC complex that is recruited by the C-terminal repressor domain of REST to actively repress genes essential to the neuronal phenotype.

Reactive oxygen species have been implicated in the pathogenesis of acute pancreatitis. Few studies have focused on the loss of endogenous antioxidants and molecular oxidative damage. Two acute pancreatitis models in rats; taurocholate (3% intraductal infusion) and cerulein (10 μg/kg/h), were used to study markers of oxidative stress: Glutathione, ascorbic acid, and their oxidized forms (glutathione disulfide and dehydroascorbic acid), malondialdehyde, and 4-hydroxynoneal in plasma and pancreas, as well as 7-hydro-8-oxo-2′-deoxyguanosine in pancreas. In both models, pancreatic glutathione depleted by 36–46% and pancreatic ascorbic acid depleted by 36–40% (p < .05). In the taurocholate model, plasma glutathione was depleted by 34% (p < .05), but there were no significant changes in plasma ascorbic acid or in plasma and pancreas dehydroascorbic acid, malondialdehyde, and 4-hydroxynoneal, and no significant changes in the pancreas glutathione disulfide/glutathione ratio. While pancreas glutathione disulfide/glutathione ratio increased in the cerulein model, there were no significant changes in plasma glutathione, plasma, or pancreas ascorbic acid, dehydroascorbic acid, 4-hydroxynoneal, and malondialdehyde, or in pancreas 7-hydro-8-oxo-2′-deoxyguanosine. Reactive oxygen species have a minor role in the intermediate stages of pancreatitis models.

Glutathione (GSH; L-gamma-glutamyl-L-cysteineglycine) is found in almost all mammalian cells, and liver has very high intracellular levels of GSH. It has many cellular functions, such as being a coenzyme, maintaining thiol/disulfide status, protection against toxic compounds and oxidative stress. GSH levels have been reported to be low in a number of pathological conditions; thus methods for increasing GSH levels are desirable. GSH may be increased by supplying its amino acid precursor cysteine, in the form of prodrugs, such as N-acetylcysteine (NAC) and 2-oxothiazolidine-4-carboxylate (OTC). It may also be increased by giving gamma-glutamylcysteine, a dipeptide precursor GSH monoester and GSH diester are effective GSH delivery drugs. Such compounds may be therapeutically useful. Gene therapy may be useful for longer term therapy of GSH deficiency.

The tumor suppressor p53 has two DNA binding domains: a central sequence-specific domain and a C-terminal sequence-independent domain. Here, we show that binding of large but not small DNAs by the C terminus of p53 negatively regulates sequence-specific DNA binding by the central domain. Four previously described mechanisms for activation of specific DNA binding operate by blocking negative regulation. Deletion of the C terminus of p53 activates specific DNA binding only in the presence of large DNA. Three activator molecules (a small nucleic acid, a monoclonal antibody against the p53 C terminus, and a C-terminal peptide of p53) stimulate sequence-specific DNA binding only in the presence of both large DNA and p53 with an intact C terminus. Our findings argue that interactions of the C terminus of p53 with genomic DNA in vivo would prevent p53 binding to specific promoters and that cellular mechanisms to block C-terminal DNA binding would be required.

p53 accumulates after DNA damage and arrests cellular growth. These findings suggest a possible role for p53 in the cellular response to DNA damage. We have previously shown that the C terminus of p53 binds DNA nonspecifically and assembles stable tetramers. In this study, we have utilized purified segments of human and murine p53s to determine which p53 domains may participate in a DNA damage response pathway. We find that the C-terminal 75 amino acids of human or murine p53 are necessary and sufficient for the DNA annealing and strand-transfer activities of p53. In addition, both full-length wild-type p53 and the C-terminal 75 amino acids display an increased binding affinity for DNA damaged by restriction digestion, DNase I treatment, or ionizing radiation. In contrast, the central site-specific DNA-binding domain together with the tetramerization domain does not have these activities. We propose that interactions of the C terminus of p53 with damaged DNA may play a role in the activation of p53 in response to DNA damage.

Glutathione (GSH) synthetase [gamma-L-glutamyl-L-cysteine:glycine ligase (ADP-forming), EC 6.3.2.3], an enzyme present in almost all cells, catalyzes the ATP-dependent synthesis of GSH from gamma-L-glutamyl-L-cysteine and glycine. Highly purified preparations of the enzyme have been obtained from rat kidney and several lower forms. The rat kidney enzyme (M(r), 118,000), which contains approximately 2% carbohydrate, is composed of two apparently identical subunits. The cDNA encoding rat kidney GSH synthetase was isolated from a rat kidney lambda gt11 cDNA library by immunoscreening with an antibody prepared against the isolated enzyme. The cDNA contains 1905 nucleotides and an open reading frame of 1422 nucleotides coding for 474 amino acids. The cDNA has a 3' untranslated region of 439 nucleotides, which includes a poly(A) tail. The deduced amino acid sequence (M(r), 52,344) contains all five of the peptide sequences that were independently determined by Edman degradation. The cDNA was expressed in Escherichia coli. The amino acid sequence of the rat kidney enzyme has no significant similarity to that of the enzyme from E. coli and shows some similarity to those deduced for the yeast and frog enzymes. Knowledge of this amino acid sequence is expected to facilitate elucidation of the sequence of the corresponding human enzyme and to lead to studies on the biochemical mechanisms involved in human GSH synthetase deficiency as well as to development of improved methods for prenatal diagnosis of these inborn diseases.

We review the findings of Cho et al. on the crystal structure of a p53 tumor suppressor-DNA complex. The core DNA binding domain of p53 folds into a structure termed a beta-sandwich, which organizes two loops and a loop-sheet-helix structure on one surface of p53 to interact with the consensus DNA recognition sequence of p53. These structures help to explain the functions of wild-type p53 and the effects of tumor-associated mutations on p53 DNA binding, transactivation and suppression of cellular proliferation.

LARGE-dependent modification enables α-dystroglycan (α-DG) to bind to its extracellular matrix ligands. Mutations in the LARGE gene and several others involved in O-mannosyl glycan synthesis have been identified in congenital and limb-girdle muscular dystrophies that are characterized by perturbed glycosylation and reduced ligand-binding affinity of α-DG. LARGE is a bifunctional glycosyltransferase that alternately transfers xylose and glucuronic acid, thereby generating the heteropolysaccharides on α-DG that confer its ligand binding. Although the LARGE paralog LARGE2 (also referred to as GYLTL1B) has likewise been shown to enhance the functional modification of α-DG in cultured cells, its enzymatic activities have not been identified. Here, we report that LARGE2 is also a bifunctional glycosyltransferase and compare its properties with those of LARGE. By means of a high-performance liquid chromatography-based enzymatic assay, we demonstrate that like LARGE, LARGE2 has xylosyltransferase (Xyl-T) and glucuronyltransferase (GlcA-T) activities, as well as polymerizing activity. Notably, however, the pH optima of the Xyl-T and GlcA-T of LARGE2 are distinct from one another and also from those of LARGE. Our results suggest that LARGE and LARGE2 catalyze the same glycosylation reactions for the functional modification of α-DG, but that they have different biochemical properties.

Going LARGE Dystroglycan (DG) is a highly glycosylated extracellular matrix (ECM) receptor involved in a variety of physiological processes, including maintenance of skeletal muscle membrane integrity and the structure and function of the central nervous system. The like-acetylglucosaminyltransferase (LARGE) is responsible for posttranslational modifications of alpha-dystroglycan (α-DG) required for its function. Now, Inamori et al. (p. 93 ) demonstrate that LARGE is a bifunctional glycosyltransferase able to transfer xylose and glucuronic acid. These modifications allow α-DG to bind the laminin-G domain–containing ECM ligands: laminin, agrin, and neurexin.

Wild-type p53 forms tetramers and multiples of tetramers. Friedman et al. (P. N. Friedman, X. B. Chen, J. Bargonetti, and C. Prives, Proc. Natl. Acad. Sci. USA 90:3319-3323, 1993) have reported that human p53 behaves as a larger molecule during gel filtration than it does during sucrose gradient sedimentation. These differences argue that wild-type p53 has a nonglobular shape. To identify structural and oligomerization domains in p53, we have investigated the physical properties of purified segments of p53. The central, specific DNA-binding domain within murine amino acids 80 to 320 and human amino acids 83 to 323 behaves predominantly as monomers during analysis by sedimentation, gel filtration, and gel electrophoresis. This consistent behavior argues that the central region of p53 is globular in shape. Under appropriate conditions, however, this segment can form transient oligomers without apparent preference for a single oligomeric structure. This region does not enhance transformation by other oncogenes. The biological implications of transient oligomerization by this central segment, therefore, remain to be demonstrated. Like wild-type p53, the C terminus, consisting of murine amino acids 280 to 390 and human amino acids 283 to 393, behaves anomalously during gel filtration and apparently has a nonglobular shape. Within this region, murine amino acids 315 to 350 and human amino acids 323 to 355 are sufficient for assembly of stable tetramers. The finding that murine amino acids 315 to 360 enhance transformation by other oncogenes strongly supports the role of p53 tetramerization in oncogenesis. Amino acids 330 to 390 of murine p53 and amino acids 340 to 393 of human p53, which have been implicated by Sturzbecher et al. in tetramerization (H.-W. Sturzbecher, R. Brain, C. Addison, K. Rudge, M. Remm, M. Grimaldi, E. Keenan, and J. R. Jenkins, Oncogene 7:1513-1523, 1992), do not form stable tetramers under our conditions. Our findings indicate that p53 has at least two autonomous oligomerization domains: a strong tetramerization domain in its C-terminal region and a weaker oligomerization domain in the central DNA binding region of p53. Together, these domains account for the formation of tetramers and multiples of tetramers by wild-type p53. The tetramerization domain is the major determinant of the dominant negative phenotype leading to transformation by mutant p53s.

Results from in vitro experiments suggest that development of nitrate tolerance is due to a depletion of vascular thiol compounds (ie, cysteine and glutathione [GSH]) necessary for the bioconversion of organic nitrates. However, it is unknown whether in vivo tolerance development is associated with changes in thiol levels. This study measures plasma and vessel tissue GSH and cysteine levels in nontolerant rats, nitrate-tolerant rats, and rats treated with the two characteristically different thiol donors N-acetyl-L-cysteine and L-2-oxothiazolidine-4-carboxylic acid (OXO). Chronically catheterized conscious rats received an intravenous infusion of either nitroglycerin (NTG, 0.2 mg/h) or matching placebo for 3 days. At day 3, the hypotensive effect of 2.5 mg NTG/kg was decreased by 74 +/- 6% (mean +/- SEM, P < .05) in the NTG-treated group (n = 7), indicating the development of tolerance. No change in the hypotensive effect of NTG was seen in the placebo group (n = 6, P > .05). Hemodynamic tolerance is not associated with changes in aorta cysteine or GSH levels as compared with the placebo group (cysteine, 77 +/- 14 versus 57 +/- 11 [mean + SEM] nmol/g; GSH, 414 +/- 62 versus 399 +/- 89 nmol/g; P > .05). However, the increase in vascular thiol levels seen after OXO treatment in nontolerant rats is completely absent in nitrate-tolerant animals.(ABSTRACT TRUNCATED AT 250 WORDS)

The tail flick withdrawal reflex (TFR) was generated by applying graded electric current to the tail of intact and spinally transected rats. In Experiment 1, separate groups of rats were tested 1, 3, 7, 10, 14, or 21 days after spinal transection. The latency, amplitude, and magnitude of the TFR was highly related to current intensity in both intact and spinal animals. However, the TFR changed dramatically as a function of the number of days between spinalization and TFR measurement. Compared to intact controls, the current intensity at which TFR was initiated (threshold) in spinal rats was elevated 1 and 3 days after transection, did not differ at 7 and 10 days, and was reduced at 14 and 21 days. Latency of TFR in spinal rats did not differ from controls 1 day after transection, but decreased steadily thereafter. Amplitude and magnitude of TFR in spinal rats remained depressed, but did show recovery toward control levels as the interval between transection and testing increased. Changes in the TFR of spinal rats was correlated with recovery of tailpinch-elicited hindlimb withdrawal. Experiment 2 demonstrated that the dose-response curve relating systemic morphine treatment to increases in TFR thresholds was shifted to the right in chronic spinal rats. Threshold increases in both spinal and intact rats were not necessarily accompanied by changes in TFR performance. These experiments establish the segmental organization of tailshock-elicited TFR and supports its use as a measure of nociceptive transmission at spinal levels.

Background Human papillomavirus (HPV) is the most identifiable cause of head and neck squamous cell cancer (HNSCC). Compared with HPV-negative HNSCC, HPV-positive HNSCC presents at an advanced stage but with significantly better survival. We created a syngeneic mouse model of HPV-positive and HPV-negative HNSCC by transforming mouse primary tonsil epithelial cells with either HPV oncogenes or a nonantigenic RNA interference strategy that affects similar oncogenic pathways. Objectives To examine the effect of radiation therapy on HPV-positive and HPV-negative tumors in immune-competent and immune-incompetent mice and to examine responses in human cancer cell lines. Design Prospective in vivo murine model. Main Outcome Measures Survival and tumor growth. Results For human and murine transformed cell lines, HPV-positive cells were more resistant to radiation and cisplatin therapy compared with HPV-negative cells. In vivo, HPV-positive tumors were more sensitive to radiation, with complete clearance at 20 Gy, compared with their HPV-negative counterparts, which showed persistent growth. Cisplatin in vivo cleared HPV-positive tumors but not HPV-negative tumors. However, neither radiation or cisplatin therapy cured immune-incompetent mice. Adoptive transfer of wild-type immune cells into immune-incompetent mice restored HPV-positive tumor clearance with cisplatin therapy. Conclusions The HPV-positive tumors are not more curable based on increased epithelial sensitivity to cisplatin or radiation therapy. Instead, radiation and cisplatin induce an immune response to this antigenic cancer. The implications of these results may lead to novel therapies that enhance tumor eradication for HPV-positive cancers.

Contains fulltext : 80334.pdf (Publisher’s version ) (Closed access) Human papillomaviruses (HPVs) are a causative factor in over 90% of cervical and 25% of head and neck squamous cell carcinomas (HNSCCs). The C terminus of the high-risk HPV 16 E6 oncoprotein physically associates with and degrades a non-receptor protein tyrosine phosphatase (PTPN13), and PTPN13 loss synergizes with H-Ras(V12) or ErbB2 for invasive growth in vivo. Oral keratinocytes that have lost PTPN13 and express H-Ras(V12) or ErbB2 show enhanced Ras/RAF/MEK/Erk signaling. In co-transfection studies, wild-type PTPN13 inhibited Ras/RAF/MEK/Erk signaling in HEK 293 cells that overexpress ErbB2, EGFR or H-Ras(V12), whereas an enzymatically inactive PTPN13 did not. Twenty percent of HPV-negative HNSCCs had PTPN13 phosphatase mutations that did not inhibit Ras/RAF/MEK/Erk signaling. Inhibition of Ras/RAF/MEK/Erk signaling using MEK inhibitor U0126 blocked anchorage-independent growth in cells lacking PTPN13. These findings show that PTPN13 phosphatase activity has a physiologically significant role in regulating MAP kinase signaling.

The cofactor ATP stimulates the formation of T-antigen double hexamers on the simian virus 40 core origin of replication (I. A. Mastrangelo, P. V. C. Hough, J. S. Wall, M. Dodson, F. B. Dean, and J. Horwitz, Nature [London] 338:658-662, 1989). We report here the pathway for the assembly of hexamers and double hexamers on the core origin. ATP triggers the cooperative assembly of hexamers on the early and late halves of the origin even when they are completely isolated. Hexamer assembly nucleates at T-antigen recognition pentanucleotides in the early half of the origin. In intact origins, assembly of the first hexamer on the early half of the origin cooperatively stimulates the assembly of a second hexamer on the adjacent late half of the origin. Thus, monomer-monomer and hexamer-hexamer interactions of T antigen, allosterically activated by ATP, constitute two distinct types of cooperative interaction with the origin. Finally, we show that the assembly of T-antigen hexamers on isolated half origins leads to the same array of structural changes that T antigen induces in intact origins. We conclude that the origin is divided into complementary halves that each promote the assembly of functional T-antigen hexamers.

Human glutathione synthetase is responsible for catalyzing the final step in glutathione biosynthesis. It is a homodimer with a monomer subunit MW of 52 kDa. Kinetic analysis reveals a departure from linearity of the Lineweaver-Burk double reciprocal plot for the binding of gamma-glutamyl substrate, indicating cooperative binding. The measured apparent K(m) values for gamma-glutamyl-alpha-aminobutyrate (an analog of gamma-glutamyl-alpha-aminobutyrate) are 63 and 164 microM, respectively. Neither ATP (K(m) of 248 microM) nor glycine (K(m) of 452 microM) exhibits such cooperative binding behavior. Although ATP is proposed to play a key role in the sequential binding of gamma-glutamyl substrate to the enzyme, the cooperative binding of the gamma-glutamyl substrate is not affected by alterations of ATP concentration. Quantitative analysis of the kinetic results for gamma-glutamyl substrate binding gives a Hill coefficient (h) of 0.75, indicating negative cooperativity. Our studies, for the first time, show that human glutathione synthetase is an allosteric enzyme with cooperative binding for gamma-glutamyl substrate.

Glutathione (GSH) synthetase [L-gamma-glutamyl-L-cysteinyl:glycine ligase (ADP-forming), EC 6.3.2.3] catalyzes the final step in GSH biosynthesis. Mammalian glutathione synthetase is a homodimer with each subunit containing an active site. We report the detailed kinetic data for purified recombinant rat glutathione synthetase. It has the highest specific activity (11 micromol/min/mg) reported for any mammalian glutathione synthetase. The apparent K(m) values for ATP and glycine are 37 and 913 microM, respectively. The Lineweaver-Burk double reciprocal plot for gamma-glutamyl substrate binding revealed a departure from linearity indicating cooperative binding. Quantitative analysis of the kinetic results for gamma-glutamyl substrate binding gives a Hill coefficient (h) of 0. 576, which shows the negative cooperativity. Neither ATP, the other substrate involved in forming the enzyme-bound gamma-glutamyl phosphate intermediate, nor glycine, which attacks this intermediate to form GSH, exhibit any cooperativity. The cooperative binding of gamma-glutamyl substrate is not affected by ATP concentration. Thus, mammalian glutathione synthetase is an allosteric enzyme.

Several genes encoding proteins critical to the neuronal phenotype, such as the brain type II sodium channel gene, are expressed to high levels only in neurons. This cell specificity is due, in part, to long-term repression in nonneural cells mediated by the repressor protein REST/NRSF (RE1 silencing transcription factor/neural-restrictive silencing factor). We show here that CoREST, a newly identified human protein, functions as a corepressor for REST. A single zinc finger motif in REST is required for CoREST interaction. Mutations of the motif that disrupt binding also abrogate repression. When fused to a Gal4 DNA-binding domain, CoREST functions as a repressor. CoREST is present in cell lines that express REST, and the proteins are found in the same immunocomplex. CoREST contains two SANT (SW13/ADA2/NCoR/TFIIIB B) domains, a structural feature of the nuclear receptor and silencing mediator for retinoid and thyroid human receptors (SMRT)-extended corepressors that mediate inducible repression by steroid hormone receptors. Together, REST and CoREST mediate repression of the type II sodium channel promoter in nonneural cells, and the REST/CoREST complex may mediate long-term repression essential to maintenance of cell identity.

Glutathione (GSH; gamma-glutamylcysteinylglycine) is ubiquitous in mammalian and other living cells. It has several important functions, including protection against oxidative stress. It is synthesized from its constituent amino acids by the consecutive actions of gamma-glutamylcysteine synthetase and GSH synthetase. gamma-Glutamylcysteine synthetase activity is modulated by its light subunit and by feedback inhibition of the end product, GSH. Treatment with an inhibitor, buthionine sulfoximine (BSO), of gamma-glutamylcysteine synthetase leads to decreased cellular GSH levels, and its application can provide a useful experimental model of GSH deficiency. Cellular levels of GSH may be increased by supplying substrates and GSH delivery compounds. Increasing cellular GSH may be therapeutically useful.

Previous studies have shown that simian virus 40 large T antigen transforms cells by binding and inactivating suppressors of cell cycle progression and tumor formation. Here, we characterize the interactions of five temperature-sensitive T antigens with the tumor suppressor proteins pRb and p53. All five mutant T antigens bind pRb at the nonpermissive temperature with efficiencies similar to that of wild-type T antigen. A single transformation-competent mutant, with a substitution of amino acid 186, binds p53 at the nonpermissive temperature. Four transformation-defective mutants, with a substitution at T antigen position 357, 422, 427, or 438, are temperature sensitive for the binding and inactivation of p53. Our findings provide a basis for understanding the behavior of cells transformed by temperature-sensitive T antigens.

The serine residue required for catalysis of gamma-glutamyl transpeptidase was identified by site-specific mutagenesis of the conserved serine residues on the basis of sequence alignment of the light subunit of human, rat, pig and two bacterial enzymes. Recombinant human gamma-glutamyl transpeptidases with replacements of these serine residues by Ala were expressed using a baculovirus-insect cell system. Substitutions of Ala at Ser-385, -413 or -425 yielded almost fully active enzymes. However, substitutions of Ala at Ser-451 or -452 yielded enzymes that were only about 1% as active as the wild-type enzyme. Further, their double mutant is only 0.002% as active as the wild type. Kinetic analysis of transpeptidation using glycylglycine as acceptor indicates that the Vmax values of Ser-451 and -452 mutants are substantially decreased (to about 3% of the wild type); however, their Km values for L-gamma-glutamyl-p-nitroanilide as donor were only increased about 5 fold compared to that of the wild type. The double mutation of Ser-451 and -452 further decreased the Vmax value to only about 0.005% of the wild type, while this mutation produced only a minor effect (2-fold increase) on the Km value for the donor. The kinetic values for the hydrolysis reaction of L-gamma-glutamyl-p-nitroanilide in the mutants followed similar trends to those for transpeptidation. The rates of inactivation of Ser-451, -452 and their double mutant enzymes by acivicin, a potent inhibitor, were less than 1% that of the wild-type enzyme. The Ki value of the double mutant for L-serine as a competitive inhibitor of the gamma-glutamyl group is only 9 fold increased over that of the wild type, whereas the Ki for the serine-borate complex, which acts as an inhibitory transition-state analog, was more than 1,000 times higher than for the wild-type enzyme. These results suggest that both Ser-451 and -452 are located at the position able to interact with the gamma-glutamyl group and participate in catalysis, probably as nucleophiles or through stabilization of the transition state.

Glutathione monoesters in which the carboxyl group of the glycine residue is esterified were previously found, in contrast to glutathione itself, to be effectively transported into various types of cells and to be converted intracellularly into glutathione. Glutathione monoesters are thus useful for prevention of oxidative stress, certain toxicities, and for treatment of glutathione deficiency. Glutathione diethyl ester is rapidly split to the glutathione monoethyl ester by mouse plasma glutathione diester alpha-esterase activity. Thus, as expected, glutathione mono- and diesters have similar effects on cellular glutathione levels in mice. However, human plasma lacks glutathione diester alpha-esterase; thus, it became of interest to compare the transport properties of glutathione mono- and diesters in human cells. We found that human cells (erythrocytes, peripheral blood mononuclear cells, fibroblasts, ovarian tumor cells, and purified T cells) transport glutathione diethyl ester much more effectively than the corresponding monoethyl (glycyl) ester. Human cells rapidly convert glutathione diethyl ester to the monoester, whose intracellular levels rise to levels that are significantly higher than levels found after application of the monoester to the cells. High levels of the monoester provide the cells with a means of producing glutathione over a period of time. We conclude that glutathione diethyl ester is highly effective as a delivery agent for glutathione monoester, and thus for glutathione, in human cells and therefore could serve to decrease oxidative stress and toxicity. Hamster (and certain other animals) also lack plasma glutathione diester alpha-esterase and therefore would be suitable animal models. Previously reported toxicity of certain glutathione ester preparations appears to reflect the presence of impurities rather than effects of the esters.

Simian virus 40 large T antigen contains a single sequence element with an arrangement of cysteines and histidines that is characteristic of a zinc finger motif. The finger region maps from amino acids 302 through 320 and has the sequence C-302 L K C-305 I K K E Q P S H Y K Y H-317 E K H-320. Previous genetic analysis has shown that the cysteine and histidine sequences and the contiguous S H Y K Y region in the finger are important for DNA replication in vivo. We show here that representative mutations in either of these elements of the finger prevent the assembly of large T antigen into stable hexamers in vitro. These same mutations have a characteristic effect on the interaction of T antigen with the simian virus 40 core origin of replication. The mutant T antigens bind to the central pentanucleotide domain of the core origin but fail to melt the adjacent inverted repeat domain and to untwist the adenine-thymine domain. These defects would prevent the formation of a replication bubble and the initiation of DNA replication. Finger mutations have lesser effects on the helicase function of T antigen and no observable effect on binding of T antigen to the mouse p53 protein. We propose that the zinc finger region contributes to protein-protein interactions essential for the assembly of stable T-antigen hexamers at the origin of replication and that hexamers are needed for subsequent alterations in the structure of origin DNA. We cannot exclude the possibility that the zinc finger region also makes specific contacts with components of origin DNA.

Objectives To test whether an immunization strategy that targets the E6 and E7 oncoproteins (E6/E7) may be an effective means to prevent the development of human papillomavirus (HPV)–positive head and neck squamous cell cancers using an in vivo mouse model and to determine whether such a response would prevent the establishment of viral transformed cells in vivo. Design Adenoviral recombinant vaccine expressing HPV-16 E6/E7 (adenovirus 5 E6/E7, or Ad5 E6/E7) was generated. Specificity and timing of the E6/E7-specific cellular immune response was determined in vivo. Adenovirus 5 E6/E7 efficacy and route of administration required for clearance of HPV–positive tumor cells were monitored. Results We generated Ad5 E6/E7 oncoproteins. Splenocytes from mice immunized with Ad5 E6/E7 produced interferon (IFN)-γ to cells expressing E6/E7 but not to cells lacking these oncoproteins. A time course of IFN-γ response showed that E6/E7-specific IFN-γ production is significantly increased in the first 2 weeks after administration of the vaccine and is substantially maintained for up to 70 days. Inoculated mice cleared E6/E7-expressing tumor 70 days after implantation. At all dosages of vaccine, mice inoculated with Ad5 E6/E7 completely cleared E6/E7-expressing tumor cells implanted 2 weeks after either intratracheal or submucosal inoculation, with significant E6/E7-specific IFN-γ production. Conclusions Immunization with HPV–16 E6/E7 oncoproteins can be an effective method of protecting a host from E6/E7-expressing head and neck squamous cell cancers via generation of a potent immune response. Such a response may be beneficial when combined with traditional treatment such as surgery, chemotherapy, or radiotherapy, thus improving the prognosis and quality of life of patients with HPV-16–associated head and neck squamous cell cancers.

Objective To determine whether proteasome inhibition could reverse E6-mediated p53 degradation, cause selective growth inhibition, and induce apoptosis in human papillomavirus E6-transformed primary tonsil epithelial cells. Design Primary human and mouse tonsil epithelial cell lines were transformed with a retrovirus containing human papillomavirus 16 oncogenes. MG132 was used to inhibit proteasome degradation in vitro and in vivo, and biochemical assays regarding p53 and apoptosis were performed. Results In cells that express E6, proteasome inhibition with MG132 restored p53 protein levels and decreased proliferation in a dose-dependent fashion that was significantly more pronounced compared with controls. However, inhibition of proliferation occurred at a lower concentration than restoration of p53 protein expression. Also, wild-type and p53 knockout mouse tonsil epithelial cells that express E6 had near-identical inhibition of growth, suggesting that growth inhibition was p53 independent. In vivo studies did not demonstrate any growth inhibition. Conclusion The findings suggest that proteasome inhibition preferentially inhibits proliferation in cells expressing E6 through a p53-independent mechanism.

Objective To provide a manipulatable system to study the mechanism of human papillomavirus 16 (HPV16) E6 –related transformation of an epithelial cell type affected by HPV16 in humans. Design Biochemical and physiological studies of mouse tonsil epithelial cells (MTECs) transformed with HPV16 oncogenes plus H -ras in vitro and in vivo. Setting Basic research laboratory. Participants C57BL/6 mice. Interventions Transduction of the HPV16 oncogenes E6 and E7 in retroviral vectors into MTECs with isolation of multiple individual clones that expressed E6, E7 , or both alone or in conjunction with H -ras . Main Outcome Measures Growth in culture, anchorage-independent growth, and growth in immune competent, syngeneic mice. Results The MTECs that expressed E6 degraded p53 by a mechanism that is inhibited by proteasomal blockade. Although normal MTECs senesced after 20 population doublings, E6 alone or in combination with E7 was sufficient to immortalize MTECs beyond 25 population doublings, lower their population-doubling time, and permit anchorage-independent growth. However, only MTECs that express E6 plus H -ras or E 6 /E7 plus H -ras formed invasive tumors in immune competent, syngeneic mice at orthotopic intraoral and subdermal sites. Conclusions We found that HPV16 E6 and E7 alone are not sufficient for invasive growth. However, the synergistic activity of H -ras and E6 was sufficient to result in invasive growth.

In vivo, topoisomerase I cleavage sites are located predominantly on the strands of simian virus 40 DNA that are the templates for discontinuous synthesis (S.E. Porter and J.J. Champoux, Mol. Cell. Biol. 9:541-550, 1989). This arrangement of sites suggests that topoisomerase I may associate with replication complexes in unique functional orientations at replication forks. We have mapped topoisomerase I cleavage sites in the simian virus 40 origin of replication in vitro under conditions suitable for DNA replication. Numerous sites cluster in the inverted repeat and AT-rich domains at the ends of the core origin and are arranged on the same strands that are cut most frequently in vivo. We propose that cleavage at these sites would allow bidirectional extension of the replication bubble induced by T antigen within the core origin of replication early in the initiation of DNA synthesis. A mutational analysis of the topoisomerase I sites confirms the importance of positions -4 to -1 and +1 in the consensus sequence 5'-A/T-A/G-A/T-T-break-G/A-3'. Surprisingly, more distant nucleotide positions also influence topoisomerase I sites in the inverted repeat and AT-rich domains of the core origin. The effects of distant sequences could be mediated by direct interactions with topoisomerase I or by the conformation of DNA in the core origin.

Glutathione monoethyl ester (L-gamma-glutamyl-L-cysteinylglycyl ethyl ester), in contrast to glutathione itself, is effectively transported into many types of cells. The ester is converted intracellularly into glutathione. Intraperitoneal injection of 35S-labeled ester into mice was followed by rapid appearance of isotope in the glutathione of liver, kidney, spleen, pancreas, and heart; the glutathione levels of these tissues also increased. Oral administration of the ester to mice also increased cellular glutathione levels. Relatively little extracellular deesterification was found. Transport of glutathione ester into human erythrocytes and intracellular conversion to glutathione was observed. The findings suggest that the glutathione ester will be useful as a radioprotecting agent and in the prevention and treatment of toxicity due to certain foreign compounds and oxygen. The ester may be useful in experimental work on glutathione transport, metabolism, and function, and in related studies on oxygen toxicity, radiation, mutagenesis, and ageing. Methods for the preparation of glutathione monoethyl ester and several related compounds are given.

Glutathione functions in catalysis, metabolism, transport, and reductive processes and in protection of cells by destruction of free radicals, reactive oxygen intermediates, and other toxic compounds of endogenous and exogenous origin. It also functions as a storage and transport form of cysteine. Depletion of glutathione (effectively accomplished by inhibition of its synthesis) increases sensitivity to radiation and to certain toxic compounds and is of value in combination with radiation therapy or chemotherapy in situations in which cell selectivity can be achieved. Increased cellular levels of glutathione protect cells against radiation and certain toxic compounds. Glutathione levels can be increased by administration of cysteine or of glutathione, but these approaches are not entirely satisfactory. Cellular glutathione levels can be increased by supplying substrate for gamma-glutamylcysteine synthetase or for glutathione synthetase. L-2-Oxothiazolidine-4-carboxylate is well transported into many cells and is converted by 5-oxoprolinase to cysteine, a substrate of gamma-glutamylcysteine synthetase. gamma-Glutamylcysteine and related compounds are effectively transported, especially into renal cells, thus providing substrate for glutathione synthetase; higher than normal levels of glutathione can be achieved because this enzyme is not significantly inhibited by glutathione, whereas gamma-glutamylcysteine synthetase is feedback-inhibited. Derivatives of glutathione that are effectively transported into cells (glutathione itself is not) offer another means of increasing glutathione levels. The monoethyl ester of glutathione (in which the glycine carboxyl group is esterified) is well transported in vivo into liver and kidney and into cultured fibroblasts and lymphoid cells. Glutathione levels much higher than usual can be obtained by this procedure, which protects lymphoid cells against the lethal effects of irradiation and mice against acetaminophen, and which therefore may be a relatively safe way to increase cellular resistance to radiation and certain toxic compounds.

Although brain cysteine levels can be increased by administration of cysteine, treatment with this amino acid causes toxicity. L-2-Oxothiazolidine-4-carboxylate, a compound in which the thiol group is masked, is effectively transported into the mouse and rat brain. It is converted intracellularly by the action of 5-oxoprolinase into L-cysteine. Study of various regions of the rat brain (cerebellum, hypothalamus, cortex, brain stem, pons, caudate nucleus) showed that the levels of cysteine increased significantly after administration of L-2-oxothiazolidine-4-carboxylate. Glutathione levels were not increased or were only slightly increased under these conditions, reflecting the low rate of glutathione synthesis in many regions of the brain.

Glutathione metabolism and transport in the choroid plexus were probed by determining the effects of administration to rats of several compounds (buthionine sulfoximine, L-2-oxothiazolidine-4-carboxylate, L-(alpha 5,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazole acetic acid, gamma-glutamyl alanine, and glutathione monoethyl ester) on the levels of glutathione and cysteine in the cerebrospinal fluid. The findings indicate that glutathione is actively metabolized in the choroid plexus by pathways similar to those in kidney and other tissues. The level of glutathione in the cerebrospinal fluid can be decreased or increased by giving compounds that do not, under similar conditions, appreciably alter total brain levels of glutathione. Glutathione monoethyl ester is effectively transported into the cerebrospinal fluid.

Administration of gamma-glutamylcystine or of gamma-glutamylcysteine disulfide to mice leads to significantly increased levels of glutathione in the kidney as compared to controls given glutamate plus cysteine (or cystinylbisglycine). Studies with gamma-glutamylcystine selectively labeled with 35S in either the internal or external S atom indicate preferential utilization of the gamma-glutamylcysteine moiety of this compound for glutathione synthesis. Mice depleted of glutathione by treatment with buthionine sulfoximine do not significantly use the disulfides gamma-glutamylcystine or gamma-glutamylcysteine disulfide but do use gamma-glutamylcysteine for glutathione synthesis. These findings suggest a pathway in which gamma-glutamylcystine, formed by transpeptidation between glutathione and cystine, is transported and reduced by transhydrogenation with glutathione to cysteine and gamma-glutamylcysteine; the latter is used directly for glutathione synthesis. The findings show transport of gamma-glutamyl amino acids, indicate an alternative pathway of glutathione synthesis, and demonstrate a means of increasing kidney glutathione levels.

The present studies show the following: (a) The plating density of cells (V79 and GM3877) affects the experimentally determined OER values. (b) Fibroblasts depleted of cysteine and GSH are more sensitive to radiation under hypoxic conditions (under aerobic conditions the absence of cysteine or GSH has no detectable effect on radiation sensitivity). (c) Addition of GEE to V79 cells (previously depleted of GSH) leads to increased intracellular GSH levels and protects the cells against radiation under hypoxic conditions.

A direct examination of the inter-organ cycle of glutathione metabolism was made by determining glutathione levels in plasma obtained from various blood vessels of the rat. High levels of GSH were found in hepatic vein plasma, relative to arterial and systemic venous levels, reflecting translocation of GSH from the liver to the plasma. Renal vein plasma has a level that is 20% of arterial plasma indicating that the kidney removes glutathione from plasma not only by glomerular filtration (which can account for 20–30% of the glutathione removed), but also by a non-filtration mechanism. Inhibitors of γ-glutamyl transpeptidase decrease the fraction of glutathione removed by the kidney to a value approaching that filtered, indicating that the non-filtration mechanism involves γ-glutamyl transpeptidase.

Treatment of mice with various gamma-glutamyl amino acids leads to marked urinary excretion of glutathione and other gamma-glutamyl compounds. There is good correlation between the affinity of gamma-glutamyl transpeptidase for various gamma-glutamyl amino acids and the extent of glutathionuria. The findings indicate that the administered gamma-glutamyl compounds effectively compete with glutathione (exported from kidney cells and present in the glomerular filtrate) for the enzyme. The administration of certain gamma-glutamyl amino acids appears to be a specific and nontoxic procedure for in vivo inhibition of gamma-glutamyl transpeptidase that may be useful in experimental work on glutathione metabolism and function and also for treatment of certain toxicities and for modulation of the metabolism of endogenous glutathione conjugates.

Experimental modulation of cellular glutathione levels has been used to explore the role of glutathione in cadmium toxicity. Mice treated with buthionine sulfoximine [an effective irreversible inhibitor of gamma-glutamylcysteine synthetase (EC 6.3.2.2) that decreases cellular levels of glutathione markedly] were sensitized to the toxic effects of CdCl2. Mice pretreated with a sublethal dose of Cd2+ to induce metallothionein synthesis were not sensitized to Cd2+ by buthionine sulfoximine. Mice sensitized to Cd2+ by buthionine sulfoximine were protected against a lethal dose of Cd2+ by glutathione mono isopropyl ester (L-gamma-glutamyl-L-cysteinylglycylisopropyl ester), but not by glutathione. These results are in accord with studies that showed that glutathione mono esters (in contrast to glutathione) are efficiently transported into cells and converted intracellularly to glutathione. The findings indicate that intracellular glutathione functions in protection against Cd2+ toxicity, and that this tripeptide provides a first line of defense against Cd2+ before induction of metallothionein synthesis occurs. The experimental approach used here in which cellular levels of glutathione are decreased or increased seems applicable to investigation of other types of metal toxicity and of other glutathione-dependent biological phenomena.