Biomedical subjects

Edward M Johnson

Publications and source records attributed to Edward M Johnson.

17 recordsLinked to original sources

PubMed2006

Role of Pur alpha in targeting mRNA to sites of translation in hippocampal neuronal dendrites.

Using genetic inactivation in the mouse, PURA, encoding Pur alpha, is demonstrated to be essential for developmentally-timed dendrite formation in the cerebellum and hippocampus. Comparison of RNA species bound by Pur alpha prompts the hypothesis that Pur alpha functions with non-coding RNA in transport of certain mRNA molecules to sites of translation in dendrites. Pur alpha binds to human BC200 RNA, implicated in dendritic targeting, and this has homologies to 7SL RNA, implicated in compartmentalized translation. Results using hippocampal rat neurons in situ show that Pur alpha binds to BC1 RNA, implicated in dendritic targeting as a mouse counterpart of BC200, and to mRNA molecules translated in dendrites; Pur alpha is specifically located in dendrites, where it is colocalized with Map2, but not in axons, where it fails to colocalize with Ankyrin G. Pur alpha and Staufen are colocalized at dendritic sites of mRNA translation. Microtubule disruptors inhibit Pur alpha dendritic targeting and allow its mislocalization to axons. Using mouse brain, double-RNA immunoprecipitation places Pur alpha together with Staufen or FMRP on BC1 RNA and specific mRNA species in vivo. These results help define a mechanism by which Pur alpha targets specific mRNA molecules to sites of dendritic translation.

Animals↗

PubMed2006

Polyoma virus infection is a prominent risk factor for bladder carcinoma in immunocompetent individuals.

Despite various reports of BK viral (BKV) DNA sequences or proteins in tumors of the urogenital tract, there has been no study statistically linking infection by this polyoma virus (PV) to tumor development. All PV are potential transforming viruses, the large T-antigen of which interacts with tumor suppressor proteins. Here, we have performed a cross-sectional study of 3,782 patients having had urine cytologic analyses, comparing those diagnosed with PV infection with those not so diagnosed. In order to focus on immunocompetent individuals, renal transplant patients, for whom a diagnosis of PV infection followed immunosuppressive therapy, were excluded. Among the 133 immunocompetent patients diagnosed with PV infection, the most frequently occurring neoplasms were bladder carcinoma (15.8%) and prostate carcinoma (3.8%). The incidence of bladder carcinoma was sufficient to statistically establish temporality in a two-sided test, linking a prior diagnosis of PV infection to a subsequent diagnosis of bladder carcinoma (odds ratio = 3.419, P < 0.001).

BK Virus↗

PubMed2006

Expression of p53 in virally infected tubular cells in renal transplant patients with polyomavirus nephropathy.

Polyomavirus (PV) infection is associated with ureteral stenosis, hemorrhagic cystitis, and interstitial nephritis in renal transplant patients. The 3 PVs detected in human beings-BK virus, JC virus, and simian virus 40-each encode highly homologous forms of a large T antigen, a transcriptional and replicational regulatory protein. We describe immunohistochemical findings in 5 renal transplant patients who developed PV nephropathy (PVN) and a sixth patient with both PVN and PV infection of the bladder mucosa. Polyomavirus infection was confirmed by immunohistochemical detection of T antigen in kidney and bladder biopsies. We report on the expression of p53 specific to virally infected cells in all biopsies positive for T antigen. Examination of posttransplant biopsies obtained from these 6 patients before they were diagnosed with PVN revealed no expression of T antigen or p53. Accumulation of p53 in PV-infected cells may occur in response to binding of p53 by T antigen, resulting in stabilization of p53. These results provide the first evidence for intracellular actions of PV T antigen in the context of nonneoplastic diseases.

Antigens, Viral, Tumor↗

PubMed2006

Renal transplant patient with polyoma virus bladder infection and subsequent polyoma virus nephropathy.

Polyoma virus nephropathy (PVN) is a significant cause of renal allograft dysfunction in transplant patients. A 58-year-old male received a cadaveric renal transplant and 12 weeks later presented with fever, diarrhea, and dysuria. He was diagnosed with a polyoma virus infection of the bladder by a transurethral bladder biopsy. One year post-transplant, he presented with renal allograft dysfunction and was diagnosed by biopsy with PVN of the non-native kidney. The diagnosis of a polyoma virus infection was confirmed by immunoreactivity to the polyoma T-antigen. We suggest that polyoma virus infection of the bladder be included in the differential diagnosis of urinary dysfunction in post-transplant patients, as such infections might be an under-recognized comorbidity in individuals with PVN.

Antigens, Polyomavirus Transforming↗

PubMed2005

Functional interaction of Puralpha with the Cdk2 moiety of cyclin A/Cdk2.

Puralpha is a sequence-specific single-stranded nucleic acid-binding protein and a member of the highly conserved Pur family. Puralpha has been shown to colocalize with cyclin A/Cdk2 and to coimmunoprecipitate with cyclin A during S-phase. Here we show that this interaction is mediated by a specific affinity of Puralpha for Cdk2. In pull-down assays GST-Puralpha efficiently binds Cdk2 and Cdk1, binds Cdk4 less efficiently, and does not display binding to Cdk6. Puralpha stimulates several-fold the phosphorylation in vitro of histone H1 by cyclin A/Cdk2, produced from baculovirus constructs. Double chromatin immunoprecipitation using antibodies to Cdk2 and Puralpha reveals that both proteins colocalize in HeLa cells to DNA segments upstream of the c-MYC gene. Pur family member Purgamma colocalizes with Cdk2 to a specific DNA segment in this region.

Animals↗

PubMed2005

Mechanism of DNA binding and localized strand separation by Pur alpha and comparison with Pur family member, Pur beta.

Pur alpha is a single-stranded (ss) DNA- and RNA-binding protein with three conserved signature repeats that have a specific affinity for guanosine-rich motifs. Pur alpha unwinds a double-stranded oligonucleotide containing purine-rich repeats by maintaining contact with the purine-rich strand and displacing the pyrimidine-rich strand. Mutational analysis indicates that arginine and aromatic residues in the repeat region of Pur alpha are essential for both ss- and duplex DNA binding. Pur alpha binds either linearized or supercoiled plasmid DNA, generating a series of regularly spaced bands in agarose gels. This series is likely due to localized unwinding by quanta of Pur alpha since removal of Pur alpha in the gel eliminates the series and since Pur alpha binding increases the sensitivity of plasmids to reaction with potassium permanganate, a reaction specific for unwound regions. Pur alpha binding to linear duplex DNA creates binding sites for the phage T4 gp32 protein, an ss-DNA binding protein that does not itself bind linearized DNA. In contrast, Pur beta lacking the Pur alpha C-terminal region binds supercoiled DNA but not linearized DNA. Similarly, a C-terminal deletion of Pur alpha can bind supercoiled pMYC7 plasmid, but cannot bind the same linear duplex DNA segment. Therefore, access to linear DNA initially requires C-terminal sequences of Pur alpha.

Amino Acid Sequence↗

PubMed2004

Site-specific loading of an MCM protein complex in a DNA replication initiation zone upstream of the c-MYC gene in the HeLa cell cycle.

The MCM proteins participate in an orderly association, beginning with the origin recognition complex, that culminates in the initiation of chromosomal DNA replication. Among these, MCM proteins 4, 6, and 7 constitute a subcomplex that reportedly possesses DNA helicase activity. Little is known about DNA sequences initially bound by these MCM proteins or about their cell cycle distribution in the chromatin. We have determined the locations of certain MCM and associated proteins by chromatin immunoprecipitation (ChIP) in a zone of initiation of DNA replication upstream of the c-MYC gene in the HeLa cell cycle. MCM7 and its clamp-loading partner Cdc6 are highly specifically colocalized by ChIP and re-ChIP in G(1) and early S on a 198-bp segment located near the center of the initiation zone. ChIP and Re-ChIP colocalizes MCM7 and ORC1 to the same segment specifically in late G(1). MCM proteins 6 and 7 can be coimmunoprecipitated throughout the cell cycle, whereas MCM4 is reduced in the complex in late S and G(2), reappearing upon mitosis. MCM7 is not visualized by immunohistochemistry on metaphase chromosomes. MCM7 is recruited to multiple sites in chromatin in S and G(2), at which time it is not detected with ORC1. The rate of dissemination is surprisingly slow and is unlikely to be simply attributed to progression with replication forks. Results indicate sequence-specific loading of MCM proteins onto DNA in late G(1) followed by a recruitment to multiple sites in chromatin subsequent to replication.

Binding Sites↗

PubMed2004

Internalization of exogenous human immunodeficiency virus-1 protein, Tat, by KG-1 oligodendroglioma cells followed by stimulation of DNA replication initiated at the JC virus origin.

JC virus (JCV) is the etiological agent of an opportunistic brain infection, progressive multifocal leukoencephalopathy (PML), in AIDS. PML is fatal in approximately 4% of HIV-infected individuals, and although the overall incidence has fallen due to highly aggressive antiretroviral therapy (HAART), this percent has remained steady. It has been shown that the Tat protein of human immunodeficiency virus-1 (HIV-1) interacts in cells with cellular protein Puralpha. This interaction can stimulate transcription of both HIV-1 and JCV genes. HIV-1, however, infects primarily microglia and astrocytes in the brain, whereas JCV infects primarily oligodendrocytes. Although HIV-1 has been shown capable of infecting oligodendrocytes in vitro (Albright et al., 1996), no instance of viral coinfection of such cells with JCV has been reported. Tat is known to be secreted from cells in which it is made. Here we ask whether such exogenous Tat can influence JCV replication in oligodendrocytes. We find that glial cells infected with either HIV-1 or JCV are in proximity at the outer edge of PML lesions. Exogenous Tat is avidly incorporated into cultured KG-1 oligodendroglioma cells over a 72-h period and is colocalized with endogenous Puralpha both nuclear and juxtanuclear. At concentrations in the medium well below the pM range, Tat stimulates several-fold the replication in vivo of DNA initiated at the JCV origin. These results define a pathway by which a protein made by HIV-1 can directly affect the course of infection by another disease-causing virus.

Cell Line, Tumor↗

PubMed2003

Efficient one-step Suzuki arylation of unprotected halonucleosides, using water-soluble palladium catalysts.

Modification of nucleosides to give pharmaceutically active compounds, mutagenesis models, and oligonucleotide structural probes continues to be of great interest. The aqueous-phase modification of unprotected halonucleosides is reported herein. Using a catalyst derived from tris(3-sulfonatophenyl)phosphine (TPPTS) and palladium acetate, 8-bromo-2'-deoxyguanosine (8-BrdG) is coupled with arylboronic acids to give 8-aryl-2'-deoxyguanosine adducts (8-ArdG) in excellent yield in a 2:1 water:acetonitrile solvent mixture. The TPPTS ligand was found to be superior to water-soluble alkylphosphines for this coupling reaction. The coupling chemistry has been extended to 8-bromo-2'-deoxyadenosine (8-BrdA) and 5-iodo-2'-deoxyuridine (5-IdU), as well as the ribonucleosides 8-bromoguanosine and 8-bromoadenosine. Good to excellent yields of arylated adducts are obtained in all cases. With use of tri(4,6-dimethyl-3-sulfonatophenyl)phosphine (TXPTS), the Suzuki coupling of 8-BrdA and 5-IdU can be accomplished in less than 1 h at room temperature. This methodology represents an efficient and general method for halonucleoside arylation that does not require prior protection of the nucleoside.

Catalysis↗

PubMed2003

A new member of the MCM protein family encoded by the human MCM8 gene, located contrapodal to GCD10 at chromosome band 20p12.3-13.

The MCM8 protein from HeLa cells, a new member of the MCM family, co-isolates through several steps with MCM6 and MCM7, and MCM8 co-immunoprecipitates with MCM4, MCM6 and MCM7, proteins reportedly forming a helicase complex involved in initiation of DNA replication. MCM8 mRNA is expressed in placenta, lung and liver, but is also significantly expressed in adult heart, a tissue with a low percentage of proliferating cells. The MCM8 gene, consisting of 19 exons, is located contrapodal to a gene, consisting of 11 exons, encoding a homolog of the yeast GCD10 gene product. The region between these two transcription units, comprising as few as 62 bp, is TATA-less and highly GC-rich, containing multiple CpG units. MCM8 expression is altered in certain forms of neoplasia. In a case of choriocarcinoma MCM8 mRNA is aberrant, leading to expression of a protein lacking 16 amino acids. In several cases of colon adenocarcinoma MCM8 expression is greatly reduced relative to matched non-cancerous tissue. The potential helicase domain of MCM8 is different from those of other MCM proteins in that it is more homologous to canonical ATP-binding domains of other known helicases. Results suggest that MCM8 may interact with other MCM proteins to alter the function of the replicative MCM protein complex.

Adenocarcinoma↗

PubMed2003

Structure-activity relationship studies of highly selective inhibitors of the dopamine transporter: N-benzylpiperidine analogues of 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine.

A series of 4-[2-[bis(4-fluorophenyl)methoxy]ethyl-1-benzylpiperidines were examined for their ability to bind to the dopamine transporter (DAT), the serotonin transporter (SERT), and the norepinephrine transporter (NET). Binding results indicated that the presence of an electron-withdrawing group in the C(4)-position of the N-benzyl group is beneficial for binding to the DAT. Several analogues have been identified with high affinity for the DAT, up to 500-fold selectivity over the SERT and about 170-fold selectivity over the NET in binding and uptake inhibition assays.

Carrier Proteins↗

PubMed2003

Puralpha is essential for postnatal brain development and developmentally coupled cellular proliferation as revealed by genetic inactivation in the mouse.

The single-stranded DNA- and RNA-binding protein, Puralpha, has been implicated in many biological processes, including control of transcription of multiple genes, initiation of DNA replication, and RNA transport and translation. Deletions of the PURA gene are frequent in acute myeloid leukemia. Mice with targeted disruption of the PURA gene in both alleles appear normal at birth, but at 2 weeks of age, they develop neurological problems manifest by severe tremor and spontaneous seizures and they die by 4 weeks. There are severely lower numbers of neurons in regions of the hippocampus and cerebellum of PURA(-/-) mice versus those of age-matched +/+ littermates, and lamination of these regions is aberrant at time of death. Immunohistochemical analysis of MCM7, a protein marker for DNA replication, reveals a lack of proliferation of precursor cells in these regions in the PURA(-/-) mice. Levels of proliferation were also absent or low in several other tissues of the PURA(-/-) mice, including those of myeloid lineage, whereas those of PURA(+/-) mice were intermediate. Evaluation of brain sections indicates a reduction in myelin and glial fibrillary acidic protein labeling in oligodendrocytes and astrocytes, respectively, indicating pathological development of these cells. At postnatal day 5, a critical time for cerebellar development, Puralpha and Cdk5 were both at peak levels in bodies and dendrites of Purkinje cells of PURA(+/+) mice, but both were absent in dendrites of PURA(-/-) mice. Puralpha and Cdk5 can be coimmunoprecipitated from brain lysates of PURA(+/+) mice. Immunohistochemical studies reveal a dramatic reduction in the level of both phosphorylated and nonphosphorylated neurofilaments in dendrites of the Purkinje cell layer and of synapse formation in the hippocampus. Overall results are consistent with a role for Puralpha in developmentally timed DNA replication in specific cell types and also point to a newly emerging role in compartmentalized RNA transport and translation in neuronal dendrites.

Animals↗

PubMed2002

Probing triplex formation by EPR spectroscopy using a newly synthesized spin label for oligonucleotides.

Spin labels have been extensively used to study the dynamics of oligonucleotides. Spin labels that are more rigidly attached to a base in an oligonucleotide experience much larger changes in their range of motion than those that are loosely tethered. Thus, their electron paramagnetic resonance spectra show larger changes in response to differences in the mobility of the oligonucleotides to which they are attached. An example of this is 5-(2,2,5,5-tetramethyl-3-ethynylpyrrolidine-1-oxyl)-uridine (1). How ever, the synthesis of this modified DNA base is quite involved and, here, we report the synthesis of a new spin-labeled DNA base, 5-(2,2,6,6-tetramethyl-4-ethynylpiperidyl-3-ene-1-oxyl)-uridine (2). This spin label is readily prepared in half the number of steps required for 1, and yet behaves in a spectroscopically analogous manner to 1 in oligonucleotides. Finally, it is shown here that both spin labels 1 and 2 can be used to detect the formation of both double-stranded and triplex DNA.

Circular Dichroism↗

PubMed2002

Piperidine analogues of 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine (GBR 12909): high affinity ligands for the dopamine transporter.

A series of 4-[2-[bis(4-fluorophenyl)methoxy]ethylpiperidines were examined for their ability to bind to the dopamine transporter (DAT), the norepinephrine transporter, and the serotonin transporter (SERT). In particular, the role of the N-substituent on affinity and selectivity for the DAT was probed. 4-[2-[Bis(4-fluorophenyl)methoxy]ethyl-1-(2-naphthylmethyl)piperidine was found to possess subnanomolar affinity (K(i) = 0.7 nM) and good selectivity for the DAT (SERT/DAT = 323).

Carrier Proteins↗

PubMed2002

Distinct proteins encoded by alternative transcripts of the PURG gene, located contrapodal to WRN on chromosome 8, determined by differential termination/polyadenylation.

A gene encoding a new member of the Pur protein family, Purgamma, has been detected upstream of, and contrapodal to, the gene encoding the Werner syndrome helicase, Wrn, at human chromosome band 8p11-12. Both the PURG and WRN genes initiate transcription at multiple sites, the major clusters of which are approximately 90 bp apart. A segment containing this region strongly promotes transcription of a reporter gene in both directions. Both promoters are TATA-less and CAAT-less and both are positively regulated by Sp1 elements. While promoter elements for the two genes are interleaved, in the contrapodal direction, certain elements critical for each gene are distinct. Sequencing of cDNAs for Purgamma mRNA reveals that two alternative coding sequences are generated from a single gene, resulting in different Purgamma C-termini. PURG-A mRNA consists of a single intronless transcript of approximately 3 kb. PURG-B mRNA results from transcription through the PURG-A polyadenylation site and splicing out of an intron of >30 kb. In this unique example of a switch, splicing of a single intron either occurs or does not occur depending upon differential termination/polyadenylation. PURG-B is the primary PURG transcript detected in testis, but it is undetectable in all members of a normal adult tissue cDNA panel. PURG-A levels are low or undetectable in the normal tissue panel, but they are greatly elevated in all members of a tumor tissue panel. PURG-B is detected in several tumor panel members.

5' Untranslated Regions↗

PubMed2001

Coordinate effects of human immunodeficiency virus type 1 protein Tat and cellular protein Puralpha on DNA replication initiated at the JC virus origin.

JC virus (JCV) causes progressive multifocal leukoencephalopathy, a demyelinating disease in brains of individuals with AIDS. Previous work has shown that the Tat protein, encoded by human immunodeficiency virus type 1 (HIV-1), can interact with cellular protein Puralpha to enhance both TAR-dependent HIV-1 transcription and JCV late gene transcription. Tat has been shown to activate JCV transcription through interaction with Puralpha, which binds to promoter sequence elements near the JCV origin of replication. DNA footprinting has shown that Puralpha and large T-antigen cooperatively interact at several binding sites in the origin and transcriptional control region. Overexpression of Puralpha inhibits replication initiated at the JCV origin by T-antigen. In transfected glial cells Tat reversed this inhibition and enhanced DNA replication. In an in vitro replication system maximal activation by Tat, more than sixfold the levels achieved with T-antigen alone, was achieved in the presence of Puralpha. Effects of mutant Tat proteins on both activation of replication and binding to Puralpha have revealed that Cys22 exerts a conformational effect that affects both activities. The origin of an archetypal strain of JCV was less susceptible to activation of replication by Tat relative to the rearranged Mad-1 strain. These results have revealed a previously undocumented role for Tat in DNA replication and have indicated a regulatory role for JCV origin auxiliary sequences in replication and activation by Tat.

Antigens, Viral, Tumor↗

PubMedDate not supplied

The Pur protein family: clues to function from recent studies on cancer and AIDS.

The Pur protein family consists of four known members in humans, the prototype gene for which is strongly conserved throughout evolution. Several investigations have now implicated Pur alpha in pathways of inhibition of oncogenic transformation. Clues to the potential importance of Pur family members in cancer are derived from observations of genetic alterations of both Pur alpha and Pur beta in myelodysplastic syndrome progressing to acute myelogenous leukemia. A role for newly-discovered Pur gamma in neoplasia is also beginning to emerge as studies have indicated that cellular levels of two Pur gamma isoforms are elevated in certain tumors. A variety of studies have now implicated Pur alpha in development of blood cells and cells of the central nervous system. Clues to the functions of Pur alpha, a key family member, have recently been derived from studies of the interactions of HIV-1 and JC virus (JCV) in AIDS. JCV causes an opportunistic infection in the brains of certain HIV-1-infected individuals. Pur alpha can influence this viral interaction through functional associations with the Tat protein and TAR RNA of HIV-1, and with large T-antigen and DNA regulatory regions of JCV. Evidence is now strong that Pur alpha interacts with both DNA and RNA and that an important aspect of its function is to recruit regulatory proteins to specific nucleic acid sequences in processes as diverse as DNA replication, gene transcription, RNA transport in the cytoplasm and compartmentalized mRNA translation.

Acquired Immunodeficiency Syndrome↗