Showing papers by "St. Jude Children's Research Hospital published in 2000"

Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response

Abstract: PERK and IRE1 are type-I transmembrane protein kinases that reside in the endoplasmic reticulum (ER) and transmit stress signals in response to perturbation of protein folding. Here we show that the lumenal domains of these two proteins are functionally interchangeable in mediating an ER stress response and that, in unstressed cells, both lumenal domains form a stable complex with the ER chaperone BiP. Perturbation of protein folding promotes reversible dissociation of BiP from the lumenal domains of PERK and IRE1. Loss of BiP correlates with the formation of high-molecular-mass complexes of activated PERK or IRE1, and overexpression of BiP attenuates their activation. These findings are consistent with a model in which BiP represses signalling through PERK and IRE1 and protein misfolding relieves this repression by effecting the release of BiP from the PERK and IRE1 lumenal domains.

A DNA transfection system for generation of influenza A virus from eight plasmids

Abstract: We have developed an eight-plasmid DNA transfection system for the rescue of infectious influenza A virus from cloned cDNA. In this plasmid-based expression system, viral cDNA is inserted between the RNA polymerase I (pol I) promoter and terminator sequences. This entire pol I transcription unit is flanked by an RNA polymerase II (pol II) promoter and a polyadenylation site. The orientation of the two transcription units allows the synthesis of negative-sense viral RNA and positive-sense mRNA from one viral cDNA template. This pol I–pol II system starts with the initiation of transcription of the two cellular RNA polymerase enzymes from their own promoters, presumably in different compartments of the nucleus. The interaction of all molecules derived from the cellular and viral transcription and translation machinery results in the generation of infectious influenza A virus. The utility of this system is proved by the recovery of the two influenza A viruses: A/WSN/33 (H1N1) and A/Teal/HK/W312/97 (H6N1). Seventy-two hours after the transfection of eight expression plasmids into cocultured 293T and MDCK cells, the virus yield in the supernatant of the transfected cells was between 2 × 105 and 2 × 107 infectious viruses per milliliter. We also used this eight-plasmid system for the generation of single and quadruple reassortant viruses between A/Teal/HK/W312/97 (H6N1) and A/WSN/33 (H1N1). Because the pol I–pol II system facilitates the design and recovery of both recombinant and reassortant influenza A viruses, it may also be applicable to the recovery of other RNA viruses entirely from cloned cDNA.

The Pezcoller lecture: cancer cell cycles revisited.

Abstract: Genetic lesions that disable key regulators of G1 phase progression in mammalian cells are present in most human cancers. Mitogen-dependent, cyclin D-dependent kinases (cdk4 and cdk6) phosphorylate the retinoblastoma (Rb) tumor suppressor protein, helping to cancel its growth-inhibitory effects and enabling E2F transcription factors to activate genes required for entry into the DNA synthetic phase (S) of the cell division cycle. Among the E2F-responsive genes are cyclins E and A, which combine with and activate cdk2 to facilitate S phase entry and progression. Accumulation of cyclin D-dependent kinases during G1 phase sequesters cdk2 inhibitors of the Cip/Kip family, complementing the effects of the E2F transcriptional program by facilitating cyclin E-cdk2 activation at the G1-S transition. Disruption of "the Rb pathway" results from direct mutational inactivation of Rb function, by overexpression of cyclin D-dependent kinases, or through loss of p16(INK4a), an inhibitor of the cyclin D-dependent kinases. Reduction in levels of p27(Kip1) and increased expression of cyclin E also occur and carry a poor prognostic significance in many common forms of cancer. The ARF tumor suppressor, encoded by an alternative reading frame of the INK4a-ARF locus, senses "mitogenic current" flowing through the Rb pathway and is induced by abnormal growth promoting signals. By antagonizing Mdm2, a negative regulator of the p53 tumor suppressor, ARF triggers a p53-dependent transcriptional response that diverts incipient cancer cells to undergo growth arrest or apoptosis. Although ARF is not directly activated by signals that damage DNA, its loss not only dampens the p53 response to abnormal mitogenic signals but also renders tumor cells resistant to treatment by cytotoxic drugs and irradiation. Lesions in the p16--cyclin D-CDK4--Rb and ARF--Mdm2--p53 pathways occur so frequently in cancer, regardless of patient age or tumor type, that they appear to be part of the life history of most, if not all, cancer cells.

Early Alterations of the Receptor-Binding Properties of H1, H2, and H3 Avian Influenza Virus Hemagglutinins after Their Introduction into Mammals

Abstract: Interspecies transmission of influenza A viruses circulating in wild aquatic birds occasionally results in influenza outbreaks in mammals, including humans. To identify early changes in the receptor binding properties of the avian virus hemagglutinin (HA) after interspecies transmission and to determine the amino acid substitutions responsible for these alterations, we studied the HAs of the initial isolates from the human pandemics of 1957 (H2N2) and 1968 (H3N2), the European swine epizootic of 1979 (H1N1), and the seal epizootic of 1992 (H3N3), all of which were caused by the introduction of avian virus HAs into these species. The viruses were assayed for their ability to bind the synthetic sialylglycopolymers 3'SL-PAA and 6'SLN-PAA, which contained, respectively, 3'-sialyllactose (the receptor determinant preferentially recognized by avian influenza viruses) and 6'-sialyl(N-acetyllactosamine) (the receptor determinant for human viruses). Avian and seal viruses bound 6'SLN-PAA very weakly, whereas the earliest available human and swine epidemic viruses bound this polymer with a higher affinity. For the H2 and H3 strains, a single mutation, 226Q-->L, increased binding to 6'SLN-PAA, while among H1 swine viruses, the 190E-->D and 225G-->E mutations in the HA appeared important for the increased affinity of the viruses for 6'SLN-PAA. Amino acid substitutions at positions 190 and 225 with respect to the avian virus consensus sequence are also present in H1 human viruses, including those that circulated in 1918, suggesting that substitutions at these positions are important for the generation of H1 human pandemic strains. These results show that the receptor-binding specificity of the HA is altered early after the transmission of an avian virus to humans and pigs and, therefore, may be a prerequisite for the highly effective replication and spread which characterize epidemic strains.

ATM phosphorylates p95/nbs1 in an S-phase checkpoint pathway

Abstract: The rare diseases ataxia-telangiectasia (AT), caused by mutations in the ATM gene, and Nijmegen breakage syndrome (NBS), with mutations in the p95/nbs1 gene, share a variety of phenotypic abnormalities such as chromosomal instability, radiation sensitivity and defects in cell-cycle checkpoints in response to ionizing radiation1,2,3,4. The ATM gene encodes a protein kinase that is activated by ionizing radiation or radiomimetic drugs5,6, whereas p95/nbs1 is part of a protein complex that is involved in responses to DNA double-strand breaks3,7. Here, because of the similarities between AT and NBS, we evaluated the functional interactions between ATM and p95/nbs1. Activation of the ATM kinase by ionizing radiation and induction of ATM-dependent responses in NBS cells indicated that p95/nbs1 may not be required for signalling to ATM after ionizing radiation. However, p95/nbs1 was phosphorylated on serine 343 in an ATM-dependent manner in vitro and in vivo after ionizing radiation. A p95/nbs1 construct mutated at the ATM phosphorylation site abrogated an S-phase checkpoint induced by ionizing radiation in normal cells and failed to compensate for this functional deficiency in NBS cells. These observations link ATM and p95/nbs1 in a common signalling pathway and provide an explanation for phenotypic similarities in these two diseases.

Caspase 8 is deleted or silenced preferentially in childhood neuroblastomas with amplification of MYCN

Abstract: Caspase 8 is a cysteine protease regulated in both a death-receptor-dependent and -independent manner during apoptosis. Here, we report that the gene for caspase 8 is frequently inactivated in neuroblastoma, a childhood tumor of the peripheral nervous system. The gene is silenced through DNA methylation as well as through gene deletion. Complete inactivation of CASP8 occurred almost exclusively in neuroblastomas with amplification of the oncogene MYCN. Caspase 8-null neuroblastoma cells were resistant to death receptor- and doxorubicin-mediated apoptosis, deficits that were corrected by programmed expression of the enzyme. Thus, caspase 8 acts as a tumor suppressor in neuroblastomas with amplification of MYCN.

The many substrates and functions of ATM.

Abstract: As its name suggests, the ATM ? 'ataxia-telangiectasia, mutated' ? gene is responsible for the rare disorder ataxia-telangiectasia. Patients show various abnormalities, mainly in their responses to DNA damage, but also in other cellular processes. Although it is hard to understand how a single gene product is involved in so many physiological processes, a clear picture is starting to emerge. online summary

Genetic polymorphism of thiopurine methyltransferase and its clinical relevance for childhood acute lymphoblastic leukemia.

Abstract: Thiopurine methyltransferase (TPMT) catalyses the S-methylation of thiopurines, including 6-mercaptopurine and 6-thioguanine. TPMT activity exhibits genetic polymorphism, with about 1/300 inheriting TPMT deficiency as an autosomal recessive trait. If treated with standard doses of thiopurines, TPMT-deficient patients accumulate excessive thioguanine nucleotides in hematopoietic tissues, leading to severe hematological toxicity that can be fatal. However, TPMT-deficient patients can be successfully treated with a 10- to 15-fold lower dosage of these medications. The molecular basis for altered TPMT activity has been defined, with rapid and inexpensive assays available for the three signature mutations which account for the majority of mutant alleles. TPMT genotype correlates well with in vivo enzyme activity within erythrocytes and leukemic blast cells and is clearly associated with risk of toxicity. The impact of 6-mercaptopurine dose intensity is also being clarified as an important determinate of event-free survival in childhood leukemia. In addition, there are emerging data that TPMT genotype may influence the risk of secondary malignancies, including brain tumors and acute myelogenous leukemia. Ongoing studies aim to clarify the influence of TPMT on thiopurine efficacy, acute toxicity, and risk for delayed toxicity. Together, these advances hold the promise of improving the safety and efficacy of thiopurine therapy.

Balanced hemagglutinin and neuraminidase activities are critical for efficient replication of influenza a virus

Abstract: The SD0 mutant of influenza virus A/WSN/33 (WSN), characterized by a 24-amino-acid deletion in the neuraminidase (NA) stalk, does not grow in embryonated chicken eggs because of defective NA function. Continuous passage of SD0 in eggs yielded 10 independent clones that replicated efficiently. Characterization of these egg-adapted viruses showed that five of the viruses contained insertions in the NA gene from the PB1, PB2, or NP gene, in the region linking the transmembrane and catalytic head domains, demonstrating that recombination of influenza viral RNA segments occurs relatively frequently. The other five viruses did not contain insertions in this region but displayed decreased binding affinity toward sialylglycoconjugates, compared with the binding properties of the parental virus. Sequence analysis of one of the latter viruses revealed mutations in the hemagglutinin (HA) gene, at sites in close proximity to the sialic acid receptor-binding pocket. These mutations appear to compensate for reduced NA function due to stalk deletions. Thus, balanced HA-NA functions are necessary for efficient influenza virus replication.

The Xenobiotic Compound 1,4-Bis(2-(3,5-Dichloropyridyloxy))Benzene Is an Agonist Ligand for the Nuclear Receptor CAR

Abstract: A wide range of xenobiotic compounds are metabolized by cytochrome P450 (CYP) enzymes, and the genes that encode these enzymes are often induced in the presence of such compounds. Here, we show that the nuclear receptor CAR can recognize response elements present in the promoters of xenobiotic-responsive CYP genes, as well as other novel sites. CAR has previously been shown to be an apparently constitutive transactivator, and this constitutive activity is inhibited by androstanes acting as inverse agonists. As expected, the ability of CAR to transactivate the CYP promoter elements is blocked by the inhibitory inverse agonists. However, CAR transactivation is increased in the presence of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), the most potent known member of the phenobarbital-like class of CYP-inducing agents. Three independent lines of evidence demonstrate that TCPOBOP is an agonist ligand for CAR. The first is that TCPOBOP acts in a dose-dependent manner as a direct agonist to compete with the inhibitory effect of the inverse agonists. The second is that TCPOBOP acts directly to stimulate coactivator interaction with the CAR ligand binding domain, both in vitro and in vivo. The third is that mutations designed to block ligand binding block not only the inhibitory effect of the androstanes but also the stimulatory effect of TCPOBOP. Importantly, these mutations do not block the apparently constitutive transactivation by CAR, suggesting that this activity is truly ligand independent. Both its ability to target CYP genes and its activation by TCPOBOP demonstrate that CAR is a novel xenobiotic receptor that may contribute to the metabolic response to such compounds.

Evolution of Swine H3N2 Influenza Viruses in the United States

Abstract: During 1998, severe outbreaks of influenza were observed in four swine herds in the United States. This event was unique because the causative agents, H3N2 influenza viruses, are infrequently isolated from swine in North America. Two antigenically distinct reassortant viruses (H3N2) were isolated from infected animals: a double-reassortant virus containing genes similar to those of human and swine viruses, and a triple-reassortant virus containing genes similar to those of human, swine, and avian influenza viruses (N. N. Zhou, D. A. Senne, J. S. Landgraf, S. L. Swenson, G. Erickson, K. Rossow, L. Liu, K.-J. Yoon, S. Krauss, and R. G. Webster, J. Virol. 73:8851–8856, 1999). Because the U.S. pig population was essentially naive in regard to H3N2 viruses, it was important to determine the extent of viral spread. Hemagglutination inhibition (HI) assays of 4,382 serum samples from swine in 23 states indicated that 28.3% of these animals had been exposed to classical swine-like H1N1 viruses and 20.5% had been exposed to the triple-reassortant-like H3N2 viruses. The HI data suggested that viruses antigenically related to the double-reassortant H3N2 virus have not become widespread in the U.S. swine population. The seroreactivity levels in swine serum samples and the nucleotide sequences of six additional 1999 isolates, all of which were of the triple-reassortant genotype, suggested that H3N2 viruses containing avian PA and PB2 genes had spread throughout much of the country. These avian-like genes cluster with genes from North American avian viruses. The worldwide predominance of swine viruses containing an avian-like internal gene component suggests that these genes may confer a selective advantage in pigs. Analysis of the 1999 swine H3N2 isolates showed that the internal gene complex of the triple-reassortant viruses was associated with three recent phylogenetically distinct human-like hemagglutinin (HA) molecules. Acquisition of HA genes from the human virus reservoir will significantly affect the efficacy of the current swine H3N2 vaccines. This finding supports continued surveillance of U.S. swine populations for influenza virus activity.

Hepatocyte migration during liver development requires Prox1

Abstract: Several genes are required during the early phases of liver specification, proliferation and differentiation1,2,3. Here we report that Prox1 is required for hepatocyte migration. Loss of Prox1 leads to formation of a smaller liver with a reduced population of clustered hepatocytes surrounded by a laminin-rich basal membrane.

H9N2 Influenza Viruses Possessing H5N1-Like Internal Genomes Continue To Circulate in Poultry in Southeastern China

Abstract: The transmission of H9N2 influenza viruses to humans and the realization that the A/Hong Kong/156/97-like (H5N1) (abbreviated HK/156/97) genome complex may be present in H9N2 viruses in southeastern China necessitated a study of the distribution and characterization of H9N2 viruses in poultry in the Hong Kong SAR in 1999. Serological studies indicated that H9N2 influenza viruses had infected a high proportion of chickens and other land-based birds (pigeon, pheasant, quail, guinea fowl, and chukka) from southeastern China. Two lineages of H9N2 influenza viruses present in the live-poultry markets were represented by A/Quail/Hong Kong/G1/97 (Qa/HK/G1/97)-like and A/Duck/Hong Kong/Y280/97 (Dk/HK/Y280/97)-like viruses. Up to 16% of cages of quail in the poultry markets contained Qa/HK/G1/97-like viruses, while about 5% of cages of other land-based birds were infected with Dk/HK/Y280/97-like viruses. No reassortant between the two H9N2 virus lineages was detected despite their cocirculation in the poultry markets. Reassortant viruses represented by A/Chicken/Hong Kong/G9/97 (H9N2) were the major H9N2 influenza viruses circulating in the Hong Kong markets in 1997 but have not been detected since the chicken slaughter in 1997. The Qa/HK/G1/97-like viruses were frequently isolated from quail, while Dk/HK/Y280/97-like viruses were predominately associated with chickens. The Qa/HK/G1/97-like viruses were evolving relatively rapidly, especially in their PB2, HA, NP, and NA genes, suggesting that they are in the process of adapting to a new host. Experimental studies showed that both H9N2 lineages were primarily spread by the aerosol route and that neither quail nor chickens showed evidence of disease. The high prevalence of quail infected with Qa/HK/G1/97-like virus that contains six gene segments genetically highly related to HK/156/97 (H5N1) virus emphasizes the need for surveillance of mammals including humans.

Latent Murine γ-Herpesvirus Infection Is Established in Activated B Cells, Dendritic Cells, and Macrophages

Abstract: Intranasal infection of mice with the murine γ-herpesvirus MHV-68 results in an acute lytic infection in the lung, followed by the establishment of lifelong latency. Development of an infectious mononucleosis-like syndrome correlates with the establishment of latency and is characterized by splenomegaly and the appearance of activated CD8+ T cells in the peripheral blood. Interestingly, a large population of activated CD8+ T cells in the peripheral blood expresses the Vβ4+ element in their TCR. In this report we show that MHV-68 latency in the spleen after intranasal infection is harbored in three APC types: B cells, macrophages, and dendritic cells. Surprisingly, since latency has not previously been described in dendritic cells, these cells harbored the highest frequency of latent virus. Among B cells, latency was preferentially associated with activated B cells expressing the phenotype of germinal center B cells, thus formally linking the previously reported association of latency gene expression and germinal centers to germinal center B cells. Germinal center formation, however, was not required for the establishment of latency. Significantly, although three cell types were latently infected, the ability to stimulate Vβ4+CD8+ T cell hybridomas was limited to latently infected, activated B cells.

Accessing complexity: the dynamics of virus-specific T cell responses.

Abstract: The cellular dynamics of the immune system are complex and difficult to measure. Access to this problematic area has been greatly enhanced by the recent development of tetrameric complexes of MHC class I glycoprotein + peptide (tetramers) for the direct staining of freshly isolated, antigen-specific CD8(+ )T cells. Analysis to date with both naturally acquired and experimentally induced infections has established that the numbers of virus-specific CD8(+) T cells present during both the acute and memory phases of the host response are more than tenfold in excess of previously suspected values. The levels are such that the virus-specific CD8(+) set is readily detected in the human peripheral blood lymphocyte compartment, particularly during persistent infections. Experimentally, it is now possible to measure the extent of cycling for tetramer (+)CD8(+) T cells during the acute and memory phases of the host response to viruses. Dissection of the phenotypic, functional, and molecular diversity of CD8(+) T cell populations has been greatly facilitated. It is hoped it will also soon be possible to analyze CD4(+) T cell populations in this way. Though these are early days and there is an enormous amount to be done, our perceptions of the shape of virus-specific cell-mediated immunity are changing rapidly.

Cooperative signals governing ARF-mdm2 interaction and nucleolar localization of the complex.

Abstract: The ARF tumor suppressor protein stabilizes p53 by antagonizing its negative regulator, Mdm2 (Hdm2 in humans). Both mouse p19ARF and human p14ARF bind to the central region of Mdm2 (residues 210 to 304), a segment that does not overlap with its N-terminal p53-binding domain, nuclear import or export signals, or C-terminal RING domain required for Mdm2 E3 ubiquitin ligase activity. The N-terminal 37 amino acids of mouse p19ARF are necessary and sufficient for binding to Mdm2, localization of Mdm2 to nucleoli, and p53-dependent cell cycle arrest. Although a nucleolar localization signal (NrLS) maps within a different segment (residues 82 to 101) of the human p14ARF protein, binding to Mdm2 and nucleolar import of ARF-Mdm2 complexes are both required for cell cycle arrest induced by either the mouse or human ARF proteins. Because many codons of mouse ARF mRNA are not recognized by the most abundant bacterial tRNAs, we synthesized ARF minigenes containing preferred bacterial codons. Using bacterially produced ARF polypeptides and chemically synthesized peptides conjugated to Sepharose, residues 1 to 14 and 26 to 37 of mouse p19ARF were found to interact independently and cooperatively with Mdm2, while residues 15 to 25 were dispensable for binding. Paradoxically, residues 26 to 37 of mouse p19ARF are also essential for ARF nucleolar localization in the absence of Mdm2. However, the mobilization of the p19ARF-Mdm2 complex into nucleoli also requires a cryptic NrLS within the Mdm2 C-terminal RING domain. The Mdm2 NrLS is unmasked upon ARF binding, and its deletion prevents import of the ARF-Mdm2 complex into nucleoli. Collectively, the results suggest that ARF binding to Mdm2 induces a conformational change that facilitates nucleolar import of the ARF-Mdm2 complex and p53-dependent cell cycle arrest. Hence, the ARF-Mdm2 interaction can be viewed as bidirectional, with each protein being capable of regulating the subnuclear localization of the other.

A triclosan-resistant bacterial enzyme

Abstract: Triclosan is an antimicrobial agent that is widely used in a variety of consumer products and acts by inhibiting one of the highly conserved enzymes (enoyl-ACP reductase, or FabI) of bacterial fatty-acid biosynthesis. But several key pathogenic bacteria do not possess FabI, and here we describe a unique triclosan-resistant flavoprotein, FabK, that can also catalyse this reaction in Streptococcus pneumoniae. Our finding has implications for the development of FabI-specific inhibitors as antibacterial agents.

Role of novel choline binding proteins in virulence of Streptococcus pneumoniae.

Abstract: The choline binding proteins (CBPs) are a family of surface proteins noncovalently bound to the phosphorylcholine moiety of the cell wall of Streptococcus pneumoniae by a conserved choline binding domain. Six new members of this family were identified, and these six plus two recently described cell wall hydrolases, LytB and LytC, were characterized for their roles in virulence. CBP-deficient mutants were constructed and tested for adherence to eukaryotic cells, colonization of the rat nasopharynx, and ability to cause sepsis. Five CBP mutants, CbpD, CbpE, CbpG, LytB, and LytC, showed significantly reduced colonization of the nasopharynx. For CbpE and -G this was attributable to a decreased ability to adhere to human cells. CbpG, a putative serine protease, also played a role in sepsis, the first observation of a pneumococcal virulence determinant strongly operative both on the mucosal surface and in the bloodstream.

Participation of ATM in insulin signalling through phosphorylation of eIF-4E-binding protein 1.

Abstract: One of the critical responses to insulin treatment is the stimulation of protein synthesis through induced phosphorylation of the eIF-4E-binding protein 1 (4E-BP1), and the subsequent release of the translation initiation factor, eIF-4E. Here we report that ATM, the protein product of the ATM gene that is mutated in the disease ataxia telangiectasia, phosphorylates 4E-BP1 at Ser 111 in vitro and that insulin treatment induces phosphorylation of 4E-BP1 at Ser 111 in vivo in an ATM-dependent manner. In addition, insulin treatment of cells enhances the specific kinase activity of ATM. Cells lacking ATM kinase activity exhibit a significant decrease in the insulin-induced dissociation of 4E-BP1 from eIF-4E. These results suggest an unexpected role for ATM in an insulin-signalling pathway that controls translation initiation. Through this mechanism, a lack of ATM activity probably contributes to some of the metabolic abnormalities, such as poor growth and insulin resistance, reported in ataxia telangiectasia cells and patients with ataxia telangiectasia.