ERAP2 functional knockout in humans does not alter surface heavy chains or HLA-B27, inflammatory cytokines or endoplasmic reticulum stress markers

Introduction Single nucleotide polymorphisms in ERAP2 are strongly associated with ankylosing spondylitis (AS). One AS-associated single nucleotide polymorphism, rs2248374, causes a truncated ERAP2 protein that is degraded by nonsense-mediated decay. Approximately 25% of the populations of European ancestry are therefore natural ERAP2 knockouts. We investigated the effect of this associated variant on HLA class I allele presentation, surface heavy chains, endoplasmic reticulum (ER) stress markers and cytokine gene transcription in AS. Methods Patients with AS and healthy controls with either AA or GG homozygous status for rs2248374 were studied. Antibodies to CD14, CD19-ECD, HLA-A-B-C, Valpha7.2, CD161, anti-HC10 and anti-HLA-B27 were used to analyse peripheral blood mononuclear cells. Expression levels of ER stress markers (GRP78 and CHOP) and proinflammatory genes (tumour necrosis factor (TNF), IL6, IL17 and IL22) were assessed by qPCR. Results There was no significant difference in HLAclass I allele presentation or major histocompatibility class I heavy chains or ER stress markers GRP78 and CHOP or proinflammatory gene expression between genotypes for rs2248374 either between cases, between cases and controls, and between controls. Discussion Large differences were not seen in HLAB27 expression or cytokine levels between subjects with and without ERAP2 in AS cases and controls. This suggests that ERAP2 is more likely to influence AS risk through other mechanisms.

Expression of glucose transporter isoforms and the insulin receptor during hamster preimplantation embryo development

During preimplantation development, embryos of many species are known to express up to five isoforms of the facilitative glucose transporter proteins (GLUT). Development of hamster blastocysts is inhibited by glucose. We therefore investigated GLUT isoform and insulin receptor (IR) expression in hamster preimplantation embryos cultured in glucose-free medium from the 8-cell stage onwards. We show that GLUT1, 3 and 8 mRNA are constitutively expressed from the 8-cell to the blastocyst stage. The IR is expressed from the morula stage onwards. Messenger RNA of the insulin-responsive GLUT4 was not detected at any stage. GLUT1 and 3 were localised by immunocytochemistry. GLUT1 was expressed in both embryoblast and trophoblast, in the latter, mainly in basal and lateral membranes directed towards the blastocoel. and embryoblast. GLUT3 was exclusively localised in the apical. membrane of trophoblast cells. We show that hamster preimplantation embryos express several GLUT isoforms thus closely resembling embryos of other mammalian species. Despite endogenous IR expression, the insulin-sensitive isoform GLUT4 was not expressed, indicating that the insulin-mediated glucose uptake known from classical insulin target cells may not be relevant for hamster blastocysts.

Expression of GC-C, a receptor-guanylate cyclase, and its endogenous ligands uroguanylin and guanylin along the rostrocaudal axis of the intestine

Members of the receptor-guanylate cyclase (rGC) family possess an intracellular catalytic domain that is regulated by an extracellular receptor domain. GC-C, an intestinally expressed rGC, was initially cloned by homology as an orphan receptor. The search for its Ligands has yielded three candidates: STa (a bacterial toxin that causes traveler's diarrhea) and the endogenous peptides uroguanylin and guanylin. Here, by performing Northern and Western blots, and by measuring [I-125]STa binding and STa-dependent elevation of cGMP levels, we investigate whether the distribution of GC-C matches that of its endogenous ligands in the rat intestine. We establish that 1) uroguanylin is essentially restricted to small bowel; 2) guanylin is very low in proximal small bowel, increasing to prominent levels in distal small bowel and throughout colon; 3) GC-C messenger RNA and STa-binding sites are uniformly expressed throughout the intestine; and 4) GC-C-mediated cGMP synthesis peaks at the proximal and distal extremes of the intestine (duodenum and colon), but is nearly absent in the middle (ileum). These observations suggest that GC-C's activity may be posttranslationally regulated, demonstrate that the distribution of GC-C is appropriate to mediate the actions of both uroguanylin and guanylin, and help to refine current hypotheses about the physiological role(s) of these peptides.

Heme is a positive regulator of cytochrome P-450 gene transcription

Inhibitors of heme biosynthesis such as CoCl2, 3-amino-1,2,4-triazole, and thioacetamide block the 3-methylcholanthrene-mediated induction of cytochrome P-450 (c + d) messenger RNAs and their transcription in rat liver. This effect is specific, since the messenger RNA levels for albumin and glutathione transferase (Ya + Yc) and their transcription are not significantly influenced under conditions of heme depletion. Exogenous administration of heme at very low doses (50 μg/100 g body wt) is able to completely counteract the effects of the heme biosynthetic inhibitors on cytochrome P-450 (c + d) messenger RNA levels and their transcription. This constitutes a direct proof for the role of heme as a positive regulator of cytochrome P-450 gene transcription.

Heme regulates cytochrome P-450 gene transcription elongation

Administration of 3-methylcholanthrene (MC) to rats results in a striking increase in the transcription of cytochrome P-450 (c+d) messenger RNA with isolated nuclei, which is blocked by the simultaneous administration of cobalt chloride, an inhibitor of heme biosynthesis. Transcription of cytochrome P-450 (c+d) mRNAs with nuclei isolated from MC treated rats shows a linear increase with time of incubation, whereas it shows a progressive decrease with incubation time in the case of nuclei isolated from MC+CoCl2 treated rats. Addition of heme in vitro (10−6M) to the latter nuclei results in a significant counteraction of the decreased cytochrome P-450 (c+d) mRNA transcription. The inhibition in transcription rates observed in MC+CoCl2 treated rat liver nuclei is more pronounced with the seventh exon probe than with the second exon probe. Once again, in vitro heme addition can counteract the inhibition observed with both the probes. Since run off transcription with isolated nuclei represents essentially elongation of the initiated transcripts, the data obtained can be interpreted on the basis that heme regulates cytochrome P-450 gene transcription elongation.

Mammalian Mat1 subunit of Transcription Factor IIH in gene-specific and general transcription

All protein-encoding genes in eukaryotes are transcribed into messenger RNA (mRNA) by RNA Polymerase II (RNAP II), whose activity therefore needs to be tightly controlled. An important and only partially understood level of regulation is the multiple phosphorylations of RNAP II large subunit C-terminal domain (CTD). Sequential phosphorylations regulate transcription initiation and elongation, and recruit factors involved in co-transcriptional processing of mRNA. Based largely on studies in yeast models and in vitro, the kinase activity responsible for the phosphorylation of the serine-5 (Ser5) residues of RNAP II CTD has been attributed to the Mat1/Cdk7/CycH trimer as part of Transcription Factor IIH. However, due to the lack of good mammalian genetic models, the roles of both RNAP II Ser5 phosphorylation as well as TFIIH kinase in transcription have provided ambiguous results and the in vivo kinase of Ser5 has remained elusive. The primary objective of this study was to elucidate the role of mammalian TFIIH, and specifically the Mat1 subunit in CTD phosphorylation and general RNAP II-mediated transcription. The approach utilized the Cre-LoxP system to conditionally delete murine Mat1 in cardiomyocytes and hepatocytes in vivo and and in cell culture models. The results identify the TFIIH kinase as the major mammalian Ser5 kinase and demonstrate its requirement for general transcription, noted by the use of nascent mRNA labeling. Also a role for Mat1 in regulating general mRNA turnover was identified, providing a possible rationale for earlier negative findings. A secondary objective was to identify potential gene- and tissue-specific roles of Mat1 and the TFIIH kinase through the use of tissue-specific Mat1 deletion. Mat1 was found to be required for the transcriptional function of PGC-1 in cardiomyocytes. Transriptional activation of lipogenic SREBP1 target genes following Mat1 deletion in hepatocytes revealed a repressive role for Mat1apparently mediated via co-repressor DMAP1 and the DNA methyltransferase Dnmt1. Finally, Mat1 and Cdk7 were also identified as a negative regulators of adipocyte differentiation through the inhibitory phosphorylation of Peroxisome proliferator-activated receptor (PPAR) γ. Together, these results demonstrate gene- and tissue-specific roles for the Mat1 subunit of TFIIH and open up new therapeutic possibilities in the treatment of diseases such as type II diabetes, hepatosteatosis and obesity.

Estradiol - 17β induces polyaromatic hydrocarbon-inducible cytochrome P-450 in chicken liver

A cDNA clone has been isolated from a chicken liver library prepared against messenger RNA isolated after chronic estradiol-17β treatment. The clone, pP-450 IA - 61, has an insert of 900nt and the sequence shows high homology to CYPIA2 subfamily from four other species. A single injection of estradiol-17β to immature chicken results in a striking induction of mRNA hybridizing to labeled pP-450IA - 61. The probe also hybridizes to mRNA induced by 3 — methylcholanthrene in chicken. These results offer direct proof for the similarity in the mode of action at the transcriptional level of polyaromatic hydrocarbons and estrogenic compounds.

The function and regulation of the U11-48K protein in U12-dependent splicing

The removal of noncoding sequences, or introns, from the eukaryotic messenger RNA precursors is catalyzed by a ribonucleoprotein complex known as the spliceosome. In most eukaryotes, two distinct classes of introns exist, each removed by a specific type of spliceosome. The major, U2-type introns account for over 99 % of all introns, and are almost ubiquitous. The minor, U12-type introns are found in most but not all eukaryotes, and reside in conserved locations in a specific set of genes. Due to their slow excision rates, the U12-type introns are expected to be involved in the regulation of the genes containing them by inhibiting the maturation of the messenger RNAs. However, little information is currently available on how the activity of the U12-dependent spliceosome itself is regulated. The levels of many known splicing factors are regulated through unproductive alternative splicing events, which lead to inclusion of premature STOP codons, targeting the transcripts for destruction by the nonsense-mediated decay pathway. These alternative splice sites are typically found in highly conserved sequence elements, which also contain binding sites for factors regulating the activation of the splice sites. Often, the activation is achieved by binding of products of the gene in question, resulting in negative feedback loops. In this study, I show that U11-48K, a protein factor specific to the minor spliceosome, specifically recognizes the U12-type 5' splice site sequence, and is essential for proper function of the minor spliceosome. Furthermore, the expression of U11-48K is regulated through a feedback mechanism, which functions through conserved sequence elements that activate alternative splicing and nonsense-mediated decay. This mechanism is conserved from plants to animals, highlighting both the importance and early origin of this mechanism in regulating splicing factors. I also show that the feedback regulation of U11-48K is counteracted by a component of the major spliceosome, the U1 small nuclear ribonucleoprotein particle, as well as members of the hnRNP F/H protein family. These results thus suggest that the feedback mechanism is finely tuned by multiple factors to achieve precise control of the activity of the U12-dependent spliceosome.

Involvement of Cytochrome P450 in Conferring Chloroquine Resistance to the Malarial Parasite, Plasmodium falciparum

The higher levels of cytochrone P-450 dependent enzyme activities reported earlier are traced to higher levels of cytochrome P-450 (CYPIIB1/B2 like) messenger RNA in the chloroquine resistant than the sensitive strains. The messenger RNA is also induced by phenobarbitone in the sensitive strain. Pretreatment with phenobarbitone affords partial protection to chloroquine toxicity in the sensitive strain and this is not due to a differential accumulation of the drug.

Isolation of transforming growth factor-beta 2 cDNA from a fish, Cyprinus carpio by RT-PCR

Transforming Growth Factors-beta (TGF-beta s) have been described in many vertebrate species of amphibians, aves and mammals. In this report we demonstrate the presence of TGF-beta 2 in pisces. TGF-beta 2 has been cloned from a fish, Cyrinus carpio, by RT-PCR using degenerate oligonucleotide primers. Sequence analysis of the amplified product and alignment of the deduced amino acid sequence with the human TGF-beta 2 amino acid sequence revealed 81% and 93% identity in the precursor and the mature regions, respectively. The northern blot analysis of fish heart RNA shows a major messenger RNA species of about 8.0 kb and two messages of very low abundance of about 5.0 kb and 4.0 kb. The identification of TGF-beta 2 isoform in Pisces and it's high degree of homology with the mammalian isoform suggests that among all TGF-beta isoforms, TGF-beta 2 is the most conserved during evolution. (C) 1997 Elsevier Science B.V.

The Cryo-EM Structure of a Complete 30S Translation Initiation Complex from Escherichia coli

11 p.

Prioritization of candidate cancer genes-an aid to oncogenomic studies

The development of techniques for oncogenomic analyses such as array comparative genomic hybridization, messenger RNA expression arrays and mutational screens have come to the fore in modern cancer research. Studies utilizing these techniques are able to highlight panels of genes that are altered in cancer. However, these candidate cancer genes must then be scrutinized to reveal whether they contribute to oncogenesis or are coincidental and non-causative. We present a computational method for the prioritization of candidate (i) proto-oncogenes and (ii) tumour suppressor genes from oncogenomic experiments. We constructed computational classifiers using different combinations of sequence and functional data including sequence conservation, protein domains and interactions, and regulatory data. We found that these classifiers are able to distinguish between known cancer genes and other human genes. Furthermore, the classifiers also discriminate candidate cancer genes from a recent mutational screen from other human genes. We provide a web-based facility through which cancer biologists may access our results and we propose computational cancer gene classification as a useful method of prioritizing candidate cancer genes identified in oncogenomic studies.

Expression of eph-family receptor tyrosine kinases and ephrins in the tadpole of the frog Xenopus laevis, and possible roles in the development of retinotectal topography

Assembling a nervous system requires exquisite specificity in the construction of neuronal connectivity. One method by which such specificity is implemented is the presence of chemical cues within the tissues, differentiating one region from another, and the presence of receptors for those cues on the surface of neurons and their axons that are navigating within this cellular environment.

Connections from one part of the nervous system to another often take the form of a topographic mapping. One widely studied model system that involves such a mapping is the vertebrate retinotectal projection-the set of connections between the eye and the optic tectum of the midbrain, which is the primary visual center in non-mammals and is homologous to the superior colliculus in mammals. In this projection the two-dimensional surface of the retina is mapped smoothly onto the two-dimensional surface of the tectum, such that light from neighboring points in visual space excites neighboring cells in the brain. This mapping is implemented at least in part via differential chemical cues in different regions of the tectum.

The Eph family of receptor tyrosine kinases and their cell-surface ligands, the ephrins, have been implicated in a wide variety of processes, generally involving cellular movement in response to extracellular cues. In particular, they possess expression patterns-i.e., complementary gradients of receptor in retina and ligand in tectum- and in vitro and in vivo activities and phenotypes-i.e., repulsive guidance of axons and defective mapping in mutants, respectively-consistent with the long-sought retinotectal chemical mapping cues.

The tadpole of Xenopus laevis, the South African clawed frog, is advantageous for in vivo retinotectal studies because of its transparency and manipulability. However, neither the expression patterns nor the retinotectal roles of these proteins have been well characterized in this system. We report here comprehensive descriptions in swimming stage tadpoles of the messenger RNA expression patterns of eleven known Xenopus Eph and ephrin genes, including xephrin-A3, which is novel, and xEphB2, whose expression pattern has not previously been published in detail. We also report the results of in vivo protein injection perturbation studies on Xenopus retinotectal topography, which were negative, and of in vitro axonal guidance assays, which suggest a previously unrecognized attractive activity of ephrins at low concentrations on retinal ganglion cell axons. This raises the possibility that these axons find their correct targets in part by seeking out a preferred concentration of ligands appropriate to their individual receptor expression levels, rather than by being repelled to greater or lesser degrees by the ephrins but attracted by some as-yet-unknown cue(s).

Mitochondria and the development of sea urchin embryos

After artificial activation or fertilization of non-nucleate fragments or eggs of the sea urchin, the mitochondria actively synthesize RNA. The RNA made in non-nucleate fragments is shown to be mostly single stranded and to be associated primarily with the low speed pellet of centrifuged cellular homogenates.

Protein synthesis is observed in non-nucleate fragments in the presence or absence of the mitochondrial RNA synthesis: it is found to be qualitatively similar but quantitatively less in the absence of the RNA synthesis. The continued syntheses of proteins in the non-nucleate fragments in the absence of mitochondrial RNA synthesis provides additional evidence for the presence of a stable messenger RNA component in the unfertilized sea urchin egg.

Since the uptake or actinomycin D was found to be inhibited by the presence of a fertilization membrane, ethidium bromide, at 10 μgs/ml, is used as an effective inhibitor of RNA synthesis in non-nucleate fragments and in early cleavage stage embryos. However, this same concentration of ethidium bromide is found to be only partially effective in blocking RNA synthesis at the mesenchyme blastula stage of development.

Low concentrations of ethidium bromide (2 and 5 μgs/ml) are found not to be lethal but to be capable of producing moderate developmental defects. In the presence of concentrations of ethidium bromide adequate to inhibit all the mitochondrial RNA synthesis (10 μgs/ml of ethidium bromide), from fertilization on, the embryos do not cleave beyond the 4-8 cell stages. When similar concentrations of ethidium bromide are added at an early mesenchyme blastula stage, the embryos do not gastrulate but continue to swim for more than 24 additional hours (adequate for control embryos to develop to a late prism stage). These results lead to the conclusion that mitochondrial RNA synthesis may be very essential for normal development to occur.

DNA is synthesized in the non-nucleate fragments of sea urchin eggs. None of the newly synthesized DNA is found in the closed circular form. When phenol extracted directly from the fragments, the DNA is found to sediment at approximately 38 and 27s in sucrose gradients but neither of these size classes could be found associated with the isolated mitochondria. The template for the synthesis of DNA in non-nucleate fragments remains unknown.

Studies on rabbit reticulocyte ribosomes and polyribosomes and their relation to hemoglobin synthesis

This dissertation is divided into three parts.

The first section is concerned with protein synthesis in cellfree systems from reticulocytes. The sub-cellular reticulocyte fractions, reagents, etc. have been examined for the presence of traces of ribonuclease, using. an assay based upon the loss of infectivity of RNA fran bacteriophage MS2. This assay is sensitive to 5 x 10^-7 γ RNase/ml. In addition, the loss of synthetic capacity of an 80S ribosome on dissociation has been studied, and can be attributed to loss of messenger RNA when the monomer is separated into subunits. The presence of ribonuclease has been shown to be a major cause of polyribosome disintegration during cell-free protein synthesis.

The second section concerns the changes in ribosomes and polyribosomes which occur during the maturation of a reticulocyte into an erythrocyte. With increasing age, the cells lose a large proportion of the ribonucleoprotein, but the percentage of ribosomes present as polyribosomes is only slightly altered. The loss of hemoglobin synthesis on maturation is probably due to both the loss of total ribosomes and to the lessened specific activity of the polyribosomes.

The third section contains analytical ultracentrifugation data on 80S ribosomes, polyribosomes, and ribosomal RNA from reticulocytes. The 60s and 40s subunits, obtained by dissociation of the 80s particle with inorganic pyrophosphate, were also studied. The RNA from reticulocyte ribosomes has been examined under a variety of denaturing conditions, including dimethyl sulfoxide treatment, formaldehyde reaction and thermal denaturation. From these studies we can conclude that the 28S and 16S RNA's are single polynucleotide chains and are not made up of smaller RNA subunits hydrogen-bonded together.