Posttranslational modification at the N terminus of the human adenovirus type 12 E1A 235R tumor antigen.

The adenovirus E1A transforming region, which encodes immortalization, partial cell transformation, and gene activation functions, expresses two early mRNAs, 13S and 12S. Multiple-T antigen species with different electrophoretic mobilities are formed from each mRNA, presumably by unknown posttranslational modifications. The adenovirus type 12 (Ad12) 13S and 12S mRNAs encode E1A T antigens of 266 and 235 amino acid residues (266R and 235R), respectively. To study possible posttranslational processing at the N and C termini and to distinguish between the Ad12 266R and 235R T antigens, we prepared antibodies targeted to synthetic peptides encoded at the common C (peptide 204) and N (peptide 202) termini of the 266R and 235R T antigens and at the unique internal domain of the 266R T antigen (peptide 206). The specificity of each anti-peptide antibody was confirmed by immunoprecipitation of the 266R and 235R T antigens produced in Escherichia coli. Immunoprecipitation analysis of the E1A T antigens synthesized in Ad12-infected KB cells revealed the following. Antibody to the common C terminus recognized three T antigens with apparent Mrs of 43,000, 42,000, and 39,000 (43K, 42K, and 39K). All three forms were phosphorylated and were present in both the nucleus and the cytoplasm. The 43K and 42K T antigens were rapidly synthesized during a 10-min pulse with [35S]methionine in Ad12-infected cells. The 43K T antigen had a half-life of 20 min, the 42K T antigen had a longer half-life of about 40 min, and the 39K T antigen became the predominant E1A T antigen. Antibodies to the unique region immunoprecipitated the 43K T antigen but not the 42K and 39K T antigens. Antibody to the N terminus immunoprecipitated the 43K and 42K T antigens but not the 39K T antigen, suggesting that the 39K T antigen possessed a modified N terminus. Partial N-terminal amino acid sequence analysis showed that the 43K and 42K T antigens contain methionine at residues 1 and 5, as predicted from the DNA sequence, whereas no methionine was released from the 39K T antigen during the first six cycles of Edman degradation. We propose that the short-lived 43K T antigen is the primary product of the 13S mRNA, the 266R T antigen; the somewhat more stable 42K T antigen is the primary product of the 12S mRNA, the 235R T antigen.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of adenovirus 12-encoded E1A tumor antigens synthesized in infected and transformed mammalian cells and in Escherichia coli

A 16-amino acid peptide, H2N-Arg-Glu-Gln-Thr-Val-Pro-Val-Asp-Leu-Ser-Val-Lys-Arg-Pro-Arg-Cys-COOH (peptide 204), targeted to the common C-terminus of human adenovirus 12 (Ad12) tumor antigens encoded by the E1A 13S mRNA and 12S mRNA, has been synthesized. Antibody prepared in rabbits against peptide 204 immunoprecipitated two proteins of apparent Mr 47,000 and 45,000 from extracts of [35S]methionine-labeled Ad12-early infected KB cells and a 47,000 protein from extracts of the Ad12-transformed hamster cell line, HE C19. Immunoprecipitation analysis of infected and transformed cells labeled with 32Pi showed that both major Ad12 E1A T antigens are phosphoproteins. Immunofluorescence microscopy of Ad12-early infected KB cells with antipeptide antibody showed the site of E1A protein concentration to be predominantly nuclear. E1A proteins were detected by immunofluorescence at 4 to 6 h postinfection and continued to increase until at least 18 h postinfection. Antipeptide 204 antibody was used to analyze the proteins synthesized in Escherichia coli cells transformed by plasmids containing cDNA copies of the Ad12 E1A 13S mRNA or 12S mRNA under the control of the tac promoter (D. Kimelman, L. A. Lucher, M. Green, K. H. Brackmann, J. S. Symington, and M. Ptashne, Proc. Natl. Acad. Sci. U.S.A., in press). A major protein of ca. 47,000 was immunoprecipitated from extracts of each transformed E. coli cell clone. Two-dimensional gel electrophoretic analysis of immunoprecipitates revealed that the T antigens synthesized in infected KB cells, transformed hamster cells, and transformed E. coli cells possess very similar molecular weights and acidic isoelectric points of 5.2 to 5.4.     

Pneumocystis jirovecii pneumonia in developing countries

Pneumocystis pneumonia (PcP) is a serious fungal infection among immunocompromised patients. In developed countries, the epidemiology and clinical spectrum of PcP have been clearly defined and well documented. However, in most developing countries, relatively little is known about the prevalence of pneumocystosis. Several articles covering African, Asian and American countries were reviewed in the present study. PcP was identified as a frequent opportunistic infection in AIDS patients from different geographic regions. A trend to an increasing rate of PcP was apparent in developing countries from 2002 to 2010.     

The adiponectin receptor homologs in C. elegans promote energy utilization and homeostasis

Adiponectin is an adipokine with insulin-sensitising actions in vertebrates. Its receptors, AdipoR1 and AdipoR2, are PAQR-type proteins with 7-transmembrane domains and topologies reversed that of GPCR's, i.e. their C-termini are extracellular. We identified three adiponectin receptor homologs in the nematode C. elegans, named paqr-1, paqr-2 and paqr-3. These are differently expressed in the intestine (the main fat-storing tissue), hypodermis, muscles, neurons and secretory tissues, from which they could exert systemic effects. Analysis of mutants revealed that paqr-1 and -2 are novel metabolic regulators in C. elegans and that they act redundantly but independently from paqr-3. paqr-2 is the most important of the three paqr genes: mutants grow poorly, fail to adapt to growth at low temperature, and have a very high fat content with an abnormal enrichment in long (C20) poly-unsaturated fatty acids when combined with the paqr-1 mutation. paqr-2 mutants are also synthetic lethal with mutations in nhr-49, sbp-1 and fat-6, which are C. elegans homologs of nuclear hormone receptors, SREBP and FAT-6 (a Δ9 desaturase), respectively. Like paqr-2, paqr-1 is also synthetic lethal with sbp-1. Mutations in aak-2, the C. elegans homolog of AMPK, or nhr-80, another nuclear hormone receptor gene, suppress the growth phenotype of paqr-2 mutants, probably because they restore the balance between energy expenditure and storage. We conclude that paqr-1 and paqr-2 are receptors that regulate fatty acid metabolism and cold adaptation in C. elegans, that their main function is to promote energy utilization rather than storage, and that PAQR class proteins have regulated metabolism in metazoans for at least 700 million years.     

Haemophilia A: from mutation analysis to new therapies

Haemophilia is caused by hundreds of different mutations and manifests itself in clinical conditions of varying severity. Despite being inherited in monogenic form, the clinical features of haemophilia can be influenced by other genetic factors, thereby confounding the boundary between monogenic and multifactorial disease. Unlike sufferers of other genetic diseases, haemophiliacs can be treated successfully by intravenous substitution of coagulation factors. Haemophilia is also the most attractive model for developing gene-therapy protocols, as the normal life expectancy of haemophiliacs allows the side effects of gene therapy, as well as its efficiency, to be monitored over long periods.     

MGluRs regulate the expression of neuronal calcium sensor proteins NCS-1 and VILIP-1 and the immediate early gene arg3.1/arc in the hippocampus in vivo

The metabotropic glutamate receptor (mGluR) agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG) is involved in several forms of hippocampal synaptic plasticity. DHPG application can induce slow-onset potentiation, a form of long-term potentiation (LTP), in the dentate gyrus and in the CA1 region in vivo. The induction of LTP correlates with increased expression levels of neuronal calcium sensor (NCS), considered as key elements for plasticity. In this study we investigated mGluR- and time-dependent changes in the expression of two different NCS proteins. Following DHPG application in vivo NCS-1 and VILIP-1 expression increased, with significant levels reached after 8 and 24h. The effect was attenuated by treatment with the group I mGluR specific antagonist S-4-carboxyphenylglycine. The immediate early gene (IEG) arg3.1/arc showed highest expression levels 2h after DHPG-treatment. Therefore, mGluRs at concentrations which induce synaptic plasticity regulate the expression of IEGs and NCS proteins in different time frames and thus contribute to late phases of synaptic plasticity.     

Activation of the sodium/hydrogen exchanger via the fibronectin-integrin pathway results in hematopoietic stimulation

The proliferative response of hematopoietic cells is regulated by many factors, including the presence and type of growth factors, the cellular microenvironment, and the physiochemical conditions prevailing in the tissue milieu. A process fundamental to all cells is the regulation of the intracellular acid-base conditions. One of the mechanisms by which intracellular pH (pH_i) is regulated is through the sodium/hydrogen exchanger, a ubiquitous membrane protein which exploits the intra- and extracellular sodium ion gradient to drive hydrogen ions out of the cell. However, activation of the exchanger via mitogenic and nonmitogenic signals leads to an increase in pH_i which, in turn, may directly or indirectly result in a proliferative response. It has been shown that interaction of fibronectin with its integrin receptor subunits α₄ and α₅ can result in activation of the Na⁺/H⁺ exchanger. In this report, we demonstrate that when mouse bone marrow cells are physically brought together in a preculture system we designate as high cell density culture (HCDC), in a small volume and at the same cellularity as that in the marrow, hematopoietic stem and progenitor cell populations are stimulated with no additional stimulation in the presence of growth factors. Neutralizing antibodies to the growth factors added to HCDC had little, if any, effect on the degree of stimulation. However, when antibodies to fibronectin or the α₄ integrin subunit were added to HCDC, inhibition was observed, indicating that the observed hematopoietic stimulation occurred via the fibronectin-integrin pathway. Addition of 5 μM 5-(N,N-hexamethylene) amiloride (5-HMA), a specific inhibitor of the Na⁺/H⁺ exchanger, also resulted in inhibition of in vitro hematopoiesis. Since the exchanger was implicated, we then measured the pH_i of normal and HCDC-treated bone marrow cells in the absence and presence of 5-HMA by flow cytometry using the fluorescent pH-sensitive indicator, carboxy SNARF-1 AM. It was found that cells subjected to HCDC exhibited a higher pH_i than normal fresh cells. In each case, the pH_i was lowered in the presence of 5-HMA. Furthermore, addition of antibodies to fibronectin or the α₄ integrin subunit to HCDC also reduced the pH_i to a similar level to that found for 5-HMA. Our results demonstrate, for the first time, that a hematopoietic stem and progenitor cell proliferative response can be initiated by activation of the Na⁺/H⁺ exchanger, leading to an increase in pH_i, via cell-cell interaction through the fibronectin-integrin pathway. This pathway could, therefore, be significant not only in normal hematopoietic regulation, but also under pathophysiological conditions. J. Cell. Physiol. 177:109–122, 1998. © 1998 Wiley-Liss, Inc.