Changing genes; losing lactase.
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Gene Expression Regulation, Enzymologic↗
Biomedical subjects
R J Grand
Publications and source records attributed to R J Grand.
Recombinant adenovirus coexpressing covalent peptide/MHC class II complex and B7-1: in vitro and in vivo activation of myelin basic protein-specific T cells.
Previous studies have demonstrated that an MHC class II molecule with an antigenic peptide genetically fused to its beta-chain is capable of presenting this peptide to CD4(+) T cells. We hypothesized that covalent peptide/class II complex may direct the accessory molecules to exert their function specifically onto T cells in a TCR-guided fashion. To test this hypothesis, we generated several recombinant adenoviruses expressing covalent myelin basic protein peptide/I-A(u) complex (MBP(1-11)/I-A(u)) and the costimulatory molecule B7-1. Functional studies demonstrated that adenovirus-infected cells are capable of activating an MBP(1-11)-specific T cell hybridoma. Coexpression of the B7-1 molecule and MBP(1-11)/I-A(u) by the same adenovirus leads to synergy in T cell activation elicited by virus-infected cells. Furthermore, studies in syngeneic mice infected with the various adenoviruses revealed that MBP(1-11)-specific T cells are specifically activated by the coexpression of B7-1 and MBP(1-11)/I-A(u) in vivo. In conclusion, the coexpression of the covalent peptide/class II complex and accessory molecules by the same adenovirus provides a unique strategy to modulate the epitope-specific T cell response in a TCR-guided fashion. This approach may be applicable to investigate the roles of other accessory molecules in the engagement of the TCR class II molecule by substituting B7-1 with other accessory molecules in the recombinant adenovirus.
Adenoviridae↗
Structural determinants outside the PXDLS sequence affect the interaction of adenovirus E1A, C-terminal interacting protein and Drosophila repressors with C-terminal binding protein.
C-Terminal binding protein (CtBP) interacts with a highly conserved amino acid motif (PXDLS) at the C terminus of adenovirus early region 1A (AdE1A) protein. This amino acid sequence has recently been demonstrated in the mammalian protein C-terminal interacting protein (CtIP) and a number of Drosophila repressors including Snail, Knirps and Hairy. In the study described here we have examined the structures of synthetic peptides identical to the CtBP binding sites on these proteins using NMR spectroscopy. It has been shown that peptides identical to the CtBP binding site in CtIP and at the N terminus of Snail form a series of beta-turns similar to those seen in AdE1A. The PXDLS motif towards the C terminus of Snail forms an alpha-helix. However, the motifs in Knirps and Hairy did not adopt well-defined structures in TFE/water mixtures as shown by the absence of medium range NOEs and a high proportion of signal overlap. The affinities of peptides for Drosophila and mammalian CtBP were compared using enzyme-linked immunosorbent assay. CtIP, Snail (N-terminal peptide) and Knirps peptides all bind to mammalian CtBP with high affinity (K(i) of 1.04, 1.34 and 0.52 microM, respectively). However, different effects were observed with dCtBP, most notably the affinity for the Snail (N-terminal peptide) and Knirps peptides were markedly reduced (K(i) of 332 and 56 microM, respectively) whilst the Hairy peptide bound much more strongly (K(i) for dCtBP of 6.22 compared to 133 microM for hCtBP). In addition we have shown that peptides containing identical PXDLS motifs but with different N and C terminal sequences have appreciably different affinities for mammalian CtBP and different structures in solution. We conclude that the factors governing the interactions of CtBPs with partner proteins are more complex than simple possession of the PXDLS motif. In particular the overall secondary structures and amino acid side chains in the binding sites of partner proteins are of importance as well as possible global structural effects in both members of the complex. These data are considered evidence for a multiplicity of CtBPs and partner proteins in the cell.
Adenovirus E1A Proteins↗
Differential activation of intestinal gene promoters: functional interactions between GATA-5 and HNF-1 alpha.
The effects of GATA-4, -5, and -6, hepatocyte nuclear factor-1 alpha (HNF-1 alpha) and -beta, and Cdx-2 on the rat and human lactase-phlorizin hydrolase (LPH) and human sucrase-isomaltase (SI) promoters were studied using transient cotransfection assays in Caco-2 cells. GATA factors and HNF-1 alpha were strong activators of the LPH promoters, whereas HNF-1 alpha and Cdx-2 were strong activators of the SI promoter, although GATA factors were also necessary for maximal activation of the SI gene. Cotransfection of GATA-5 and HNF-1 alpha together resulted in a higher activation of all three promoters than the sum of the activation by either factor alone, demonstrating functional cooperativity. In the human LPH promoter, an intact HNF-1 binding site was required for functional synergy. This study is the first to demonstrate 1) differential activation of the LPH and SI promoters by multiple transcription factors cotransfected singly and in combination and 2) that GATA and HNF-1 transcription factors cooperatively activate intestinal gene promoters. Synergistic activation is a mechanism by which higher levels of tissue-specific expression might be attained by overlapping expression of specific transcription factors.
Base Sequence↗
Structural determinants in adenovirus 12 E1A involved in the interaction with C-terminal binding protein 1.
The interaction between the C-terminal binding protein 1 (CtBP-1) and purified Ad12 E1A protein has been examined through the use of a combination of biophysical techniques. A fragment equivalent to the 77 C-terminal amino acids of Ad12 E1A (Ad12 77-a.a. E1A) was generated by limited proteolysis of Ad12 266-a.a. E1A at Phe(187) and/or Tyr(189) using chymotrypsin. The impact of deletion of the 189 N-terminal amino acids from E1A on the equilibrium dissociation constant K(d) for binding to CtBP was assessed using ELISA in vitro binding assays and intrinsic fluorescence spectroscopy. Values of K(d) of 4.0 and 38 nM were determined for full-length and truncated forms of E1A, respectively. Circular dichroism spectroscopic studies revealed that the conformation adopted by these polypeptides is dependent on the surrounding environment, which is predominately randomly folded when free in solution, but adopting a more ordered alpha-helical secondary structure in the presence of trifluoroethanol. Using nuclear magnetic resonance (NMR) spectroscopy to examine the interaction between Ad E1A and CtBP it was observed that the chemical shift positions of individual backbone amide nitrogen atoms were well resolved in (15)N-(1)H-HSQC NMR spectra performed on samples of isotopically (15)N-labeled Ad12 77-a.a. E1A. In the presence of CtBP, signals of backbone amide nitrogen atoms displayed increased linewidth consistent with an increase in molecular mass upon binding CtBP. In addition, some signals that have been attributed to Val(254/256) and Leu(259), and reside within the binding site for CtBP on E1A, are shifted in the (15)N- and/or (1)H-dimensions, defining specific contacts between E1A and CtBP. These data suggest that structural determinants in the C-terminal PXDLS binding motif in the rest of exon 2 and in exon 1 all contribute to optimizing the conformation of the binding site on Ad12 E1A for CtBP. However, no interaction was observed between CtBP and truncated Ad12 E1A, which no longer contained the C-terminal binding motif.
Adenovirus E1A Proteins↗
Regulation of the 26S proteasome by adenovirus E1A.
We have identified the N-terminus of adenovirus early region 1A (AdE1A) as a region that can regulate the 26S proteasome. Specifically, in vitro and in vivo co-precipitation studies have revealed that the 19S regulatory components of the proteasome, Sug1 (S8) and S4, bind through amino acids (aa) 4-25 of Ad5 E1A. In vivo expression of wild-type (wt) AdE1A, in contrast to the N-terminal AdE1A mutant that does not bind the proteasome, reduces ATPase activity associated with anti-S4 immunoprecipitates relative to mock-infected cells. This reduction in ATPase activity correlates positively with the ability of wt AdE1A, but not the N-terminal deletion mutant, to significantly reduce the ability of HPV16 E6 to target p53 for ubiquitin-mediated proteasomal degradation. AdE1A/proteasomal complexes are present in both the cytoplasm and the nucleus, suggesting that AdE1A interferes with both nuclear and cytoplasmic proteasomal degradation. We have also demonstrated that wt AdE1A and the N-terminal AdE1A deletion mutant are substrates for proteasomal-mediated degradation. AdE1A degradation is not, however, mediated through ubiquitylation, but is regulated through phosphorylation of residues within a C-terminal PEST region (aa 224-238).
ATPases Associated with Diverse Cellular Activitie↗
Consequences of disruption of the interaction between p53 and the larger adenovirus early region 1B protein in adenovirus E1 transformed human cells.
The adenovirus early region 1B (Ad E1B) genes have no transforming capability of their own but markedly increase the transformation frequency of Ad E1A following co-transfection into mammalian cells. The larger E1B proteins of both Ad2/5 and Ad12 bind to p53 and inhibit its ability to transcriptionally activate other genes. We have previously demonstrated that synthetic peptides identical to the binding sites for p53 on both the Ad2 and Ad12 E1B proteins will disrupt the interaction in vivo and in vitro. In the work presented here we have examined the effects of complex dissociation on Ad E1-transformed human cells. It has been shown, using confocal microscopy, that when the peptide identical to the p53 binding site was added to Ad5 E1-transformed cells it initally located in the cytoplasmic dense bodies where it caused disruption of the p53/E1B complex. Peptide and p53 then translocated to the nucleus. In Ad12 E1-transformed cells the peptide localized in the nucleus directly and there caused a reorganization of p53 staining from a highly organized, 'flecked' distribution to one in which nuclear staining was homogeneous and diffuse. Peptides added to either Ad5 E1 or Ad12 E1 transformed cells resulted in the release of transcriptionally active p53. Interestingly, the level of p53 then fell presumably as a result of proteasomal action - this was probably a reflection of the short half-life of 'free' (i.e. dissociated) p53 compared to that of the bound protein. Free p53 did not cause apoptosis in target cells probably due to the presence of the smaller (19K) E1B proteins. However, addition of peptide leads to a significant reduction in cell growth rate. We have further demonstrated that a significant proportion of those cells which had taken up peptide had ceased DNA synthesis, probably due to a p53-induced cell cycle arrest. The role of the larger EIB protein during transformation is considered in view of these data.
Adenoviridae↗
Brush border myosin I (BBMI): a basally localized transcript in human jejunal enterocytes.
To extend our recent observation that villin mRNA, encoding an apical microvillous protein, is dichotomously localized in the basal region of human enterocytes, we examined the localization of mRNAs for brush border myosin I (BBMI) and intestinal fimbrin (I-fim). In situ hybridization indicated that BBMI mRNA localized to the basal region of human enterocytes, whereas the mRNA for I-fim distributed diffusely. To facilitate study of potential mechanisms of mRNA targeting, we cloned a full-length cDNA for BBMI including its 5'- and 3'-untranslated regions (UTRs). This cDNA shares 86% sequence identity with bovine BBMI and 85% with rat BBMI. Sequence analysis revealed no obvious similarity between the 3'-UTRs of BBMI and villin. This study provides evidence of novel sorting pathways for intestinal microvillous cytoskeletal proteins.
Base Sequence↗
The structure of the site on adenovirus early region 1A responsible for binding to TATA-binding protein determined by NMR spectroscopy.
Previous detailed mutational analysis has shown that the binding site on adenovirus (Ad) early region 1A (E1A) for TATA-binding protein (TBP) is located toward the N terminus of conserved region 3 (CR3). Here we demonstrate that synthetic peptides of between 15 and 22 amino acids, identical to amino acid sequences of CR3 present in the larger Ad5 E1A (13 S product) and in both the Ad12 E1A (13 and 12 S products) proteins that lie N-terminal to the zinc finger motif, can disrupt binding of E1A to TBP. These findings suggest that the peptides are biologically active in terms of interacting with TBP and must therefore comprise some, if not all, of the TBP binding site on E1A. The interaction between Ad12 E1A and TBP was confirmed by direct co-precipitation experiments. In 1H NMR studies of CR3 peptides, regular patterns of NOEs were observed from which their conformational preferences in aqueous solution were determined. Both Ad5 and Ad12 peptides were shown to contain regions of helical backbone structure in 50% trifluoroethanol. In each case, the type and intensities of NOE cross-peaks observed correlated best to alpha-helical turns. These helices are more extensive in larger peptides and extend from Glu141 to Val147 and from Arg144 to Pro152 in the full-length Ad5 and Ad12 13S E1A proteins, respectively. The structure of a 19-residue Ad5 CR3 peptide carrying the V147L mutation in the full-length protein that abolishes TBP binding was examined. No significant differences between the substituted and wild type peptides were observed, suggesting that this substitution in the intact protein may cause disruption of global rather than local structures.
Adenovirus E1A Proteins↗
Definition of a major p53 binding site on Ad2E1B58K protein and a possible nuclear localization signal on the Ad12E1B54K protein.
Previous studies have established that adenovirus 2/5 early region 1B (Ad E1B) 58K protein binds p53 strongly and co-localizes with it to cytoplasmic dense bodies whilst the homologous Ad12E1B54K protein binds only weakly and co-localizes primarily to the nucleus in Ad12E1 transformed cells. We have used these properties of the E1B proteins from different viral serotypes to map the p53 binding site on the Ad2/5 protein. A set of chimaeric genes was constructed containing different proportions of the Ad12 and Ad2E1B DNA. These, together with Ad12E1A and E1B19K DNA, were transfected into baby rat kidney cells and transformed lines isolated. From an examination of the properties of these Ad12/Ad2E1B fusion proteins in co-immunoprecipitation and subcellular localization experiments it has been concluded that the p53 binding site on Ad2E1B58K protein lies between amino acids 216 and 235 and that the homologous region on Ad12E1B54K protein also binds p53. In addition, a unique nuclear localization signal is located on Ad12E1B54K between residues 228 and 239. We suggest that primary structure differences in these regions of the Ad2 and Ad12E1B proteins are responsible for the different subcellular localizations in AdE1 transformants.
Adenoviridae↗
Adenovirus early region 1A protein binds to mammalian SUG1-a regulatory component of the proteasome.
Adenovirus early region 1A (Ad E1A) is a multifunctional protein which is essential for adenovirus-mediated transformation and oncogenesis. Whilst E1A is generally considered to exert its influence on recipient cells through regulation of transcription it also increases the level of cellular p53 by increasing the protein half-life. With this in view, we have investigated the relationship of Ad E1A to the proteasome, which is normally responsible for degradation of p53. Here we have shown that both Ad5 and Ad12 E1A 12S and 13S proteins can be co-immunoprecipitated with proteasomes and that the larger Ad12 E1A protein binds strongly to at least three components of the 26S but not 20S proteasome. One of these interacting species has been identified as mammalian SUGI, a proteasome regulatory component which also plays a role in the cell as a mediator of transcription. In vitro assays have demonstrated a direct interaction between Ad12 E1A 13S protein and mouse SUGI. Following infection of human cells with Ad5 wt and Ad5 mutants with lesions in the E1A gene it has been shown that human SUG1 can be co-immunoprecipitated with full-length E1A and with E1A carrying a deletion in conserved region 1 which is the region considered to be responsible for increased expression of p53. We have concluded therefore that Ad EIA binds strongly to SUGI but that this interaction is not responsible for inhibition of proteasome activity. This is consistent with the observation that purified Ad12 E1A inhibits the activity of the purified 20S but not 26S proteasomes. We have also demonstrated that SUGI can be co-immunoprecipitated with SV40 T and therefore we suggest that this may represent a common interaction of transforming proteins of DNA tumour viruses.
ATPases Associated with Diverse Cellular Activitie↗
Development of the human gastrointestinal tract: twenty years of progress.
A combination of approaches has begun to elucidate the mechanisms of gastrointestinal development. This review describes progress over the last 20 years in understanding human gastrointestinal development, including data from both human and experimental animal studies that address molecular mechanisms. Rapid progress is being made in the identification of genes regulating gastrointestinal development. Genes directing initial formation of the endoderm as well as organ-specific patterning are beginning to be identified. Signaling pathways regulating the overall right-left asymmetry of the gastrointestinal tract and epithelial-mesenchymal interactions are being clarified. In searching for extrinsic developmental regulators, numerous candidate trophic factors have been proposed, but compelling evidence remains elusive. A critical gene that initiates pancreas development has been identified, as well as a number of genes regulating liver, stomach, and intestinal development. Mutations in genes affecting neural crest cell migration have been shown to give rise to Hirschsprung's disease. Considerable progress has been achieved in understanding specific phenomena, such as the transcription factors regulating expression of sucrase-isomaltase and fatty acid-binding protein. The challenge for the future is to integrate these data into a more complete understanding of the physiology of gastrointestinal development.
Aging↗
The fate of E- and P-cadherin during the early stages of apoptosis.
Caspases are responsible for the proteolysis of many cytoskeletal proteins in apoptotic cells. It has been demonstrated here that during cisplatin-induced apoptosis of human embryo retinoblasts both E- and P-cadherin were degraded by caspases, giving initially major polypeptide products of apparent molecular weights 48 K and 104 K respectively. This proteolysis occurred over a similar time-scale to the observed degradation of PARP and to the onset of DNA fragmentation but appreciably later than p53 induction and cleavage of Mdm2 and p21. Addition of caspase inhibitors such as Z-VAD-FMK inhibited apoptosis and cadherin degradation. Co-immunoprecipitation studies carried out on viable cells confirmed previously observed complexes between cadherins and alpha and beta catenin and between the catenins themselves. These interactions were sustained in apoptotic cells as long as the protein components remained intact. Using confocal microscopy it has been shown that cytoskeletal changes associated with apoptosis precede degradation of catenins and cadherins by several hours. In particular, after addition of cisplatin relatively rapid (within 3 h) re-localization of adherens junction proteins from the cell periphery to the cytoplasm was observed whereas little cadherin or catenin degradation occurred until 10 h. We conclude that neither caspase-mediated degradation of cytoskeletal components nor disruption of adherens junction protein-protein interactions is required for morphological change.
Apoptosis↗
Propranolol in prevention of portal hypertensive hemorrhage in children: a pilot study.
BACKGROUND: Data regarding the use of propranolol in the prevention of portal hypertensive hemorrhage in pediatric patients are limited despite its widespread use in adults with cirrhosis. The purpose of this study was to evaluate safety and efficacy of propranolol in the management of portal hypertension in the pediatric population. METHODS: Medical information was retrieved from the records of 21 children with portal hypertension who received propranolol either before or after an episode of gastrointestinal bleeding. Data collected included diagnosis, type of portal hypertension, age at initiation of therapy, bleeding episodes before and during propranolol therapy, degree of reduction of heart rate, adherence, and adverse effects. RESULTS: Fourteen of 21 patients did not experience portal hypertensive bleeding while receiving propranolol. Of the seven patients who had bleeding episodes, two had failed to adhere to the medication regimen, and four were receiving doses of less than 1 mg/kg per day. Only one of the four patients who experienced bleeding before initiation of therapy also bled while receiving propranolol, and two of the three patients who had a heart rate reduction of less than 25% each experienced a bleeding episode. Side effects were minimal and did not necessitate discontinuation of therapy in any patient. CONCLUSIONS: Propranolol was well tolerated with minimal side effects in our patients with portal hypertension. Adherence and adequacy of dosage (>1 mg/kg per day, more than twice daily dose frequency) are important determinants of efficacy. A reduction in heart rate of less than 25% may be associated with suboptimal efficacy.
Adolescent↗
Gene therapy for gastrointestinal disorders: just around the corner?
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Forecasting↗
The replicative capacities of large E1B-null group A and group C adenoviruses are independent of host cell p53 status.
Recent reports suggest that an early region 1B (E1B) 55, 000-molecular-weight polypeptide (55K)-null adenovirus type 5 (Ad5) mutant (dl1520) can replicate to the same extent as wild-type (wt) Ad5 in cells either deficient or mutated in p53, implicating p53 in limiting viral replication in vivo. In contrast, we show here that the replicative capacity of Ad5 dl1520 is wholly independent of host cell p53 status, as is the replicative capacity of comparable Ad12 E1B 54K-null adenoviruses (Ad12 dl620 and Ad12 hr703). Furthermore, we show that there is no requirement for complex formation between p53 and Ad5 E1B 55K or Ad12 E1B 54K for a productive infection, such that wt Ad5 and wt Ad12 will both replicate in cells which are null for p53. In addition, we find that these Ad5 and Ad12 mutant viruses induce S phase irrespective of the p53 status of the cell and that, therefore, S-phase induction does not correlate with the replicative capacity of the virus. Interestingly, the replicative capacities of the large E1B-null adenoviruses correlated positively with the ability to express E1B 19K and were related to the ability to repress premature adenovirus-induced apoptosis. Infection of primary human cells indicated that Ad5 dl1520, wt Ad5, and wt Ad12 replicated better in cycling normal human skin fibroblasts (HSFs) than in quiescent HSFs. Thus, the cell cycle status of the host cell, upon infection, also influences viral yield.
Adenoviridae↗
Assignment of the yeast APG5 human homologue APG5L to chromosome band 6q21 by fluorescence in situ hybridisation.
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Autophagy-Related Protein 5↗
Specific cleavage of gamma catenin by caspases during apoptosis.
Caspase-mediated proteolysis of cytoskeletal proteins during apoptosis appears to be commonplace. Enlarging on previous studies we have shown here that gamma catenin, like beta catenin, was degraded during cisplatin-induced apoptosis, initially giving a major product of 75 kDa. This truncated protein could be co-immunoprecipitated with alpha catenin. Addition of caspase inhibitors to cells in the presence of cisplatin appreciably reduced the proteolysis of gamma catenin as well as the level of apoptosis. Only limited degradation of alpha catenin was observed even at very late times when over 90% of cells in the culture were apoptotic. Immunohistochemical staining showed that during apoptosis there was a relocation of alpha, beta, and gamma catenin from the periphery of the cell to the cytoplasm, at the same time as other morphological changes commonly associated with apoptosis occurred. Interestingly, the changes in localisation of the catenins preceded proteolysis by several hours. In the presence of cisplatin and caspase inhibitor no change in distribution of catenins was observed, suggesting that re-localisation requires caspase activity but not necessarily directed against beta and gamma catenins.
Adenovirus E1A Proteins↗