bluuebunny/crossref_metadata_2025 huggingface dataset:
Adeno-Associated Virus Type 2-Mediated Transduction of Human Monocyte-Derived Dendritic Cells: Implications for Ex Vivo Immunotherapy
Authors: Ponnazhagan Selvarangan , Mahendra Gandham , Curiel David T. , Shaw Denise R.
Published: July 2002

Dendritic cells (DCs) are pivotal antigen-presenting cells for regulating immune responses. A major focus of contemporary vaccine research is the genetic modification of DCs to express antigens or immunomodulatory molecules, utilizing a variety of viral and nonviral vectors, to induce antigen-specific immune responses that ameliorate disease states as diverse as malignancy, infection, autoimmunity, and allergy. The present study has evaluated adeno-associated virus (AAV) type 2 as a vector for ex vivo gene transfer to human peripheral blood monocyte (MO)-derived DCs. AAV is a nonpathogenic parvovirus that infects a wide variety of human cell lineages in vivo and in vitro, for long-term transgene expression without requirements for cell proliferation. The presented data demonstrate that recombinant AAV (rAAV) can efficiently transduce MOs as well as DCs generated by MO culture with granulocyte-macrophage colony-stimulating factor plus interleukin in vitro. rAAV transgene expression in MO-derived DCs could be enhanced by etoposide, previously reported to enhance AAV gene expression. rAAV transduction of freshly purified MO followed by 7 days of culture with cytokines to generate DCs, and subsequent sorting for coexpression of DC markers CD1a and CD40, showed robust transgene expression as well as evidence of nuclear localization of the rAAV genome in the DC population. Phenotypic analyses using multiple markers and functional assays of one-way allogeneic mixed leukocyte reactions indicated that rAAV-transduced MO-derived DCs were as equivalent to nontransduced DCs. These results support the utility of rAAV vectors for future human DC vaccine studies.
The Genome of Turkey Herpesvirus
Authors: Afonso C. L. , Tulman E. R. , Lu Z. , Zsak L. , Rock D. L. , Kutish G. F.
Published: July 2002

Here we present the first complete genomic sequence of Marek's disease virus serotype 3 (MDV3), also known as turkey herpesvirus (HVT). The 159,160-bp genome encodes an estimated 99 putative proteins and resembles alphaherpesviruses in genomic organization and gene content. HVT is very similar to MDV1 and MDV2 within the unique long (UL) and unique short (US) genomic regions, where homologous genes share a high degree of colinearity and their proteins share a high level of amino acid identity. Within the UL region, HVT contains 57 genes with homologues found in herpes simplex virus type 1 (HSV-1), six genes with homologues found only in MDV, and two genes (HVT068 and HVT070 genes) which are unique to HVT. The HVT US region is 2.2 kb shorter than that of MDV1 (Md5 strain) due to the absence of an MDV093 (SORF4) homologue and to differences at the UL/short repeat (RS) boundary. HVT lacks a homologue of MDV087, a protein encoded at the UL/RS boundary of MDV1 (Md5), and it contains two homologues of MDV096 (glycoprotein E) in the RS. HVT RS are 1,039 bp longer than those in MDV1, and with the exception of an ICP4 gene homologue, the gene content is different from that of MDV1. Six unique genes, including a homologue of the antiapoptotic gene
Bcl-2
, are found in the RS. This is the first reported
Bcl-2
homologue in an alphaherpesvirus. HVT long repeats (RL) are 7,407 bp shorter than those in MDV1 and do not contain homologues of MDV1 genes with functions involving virulence, oncogenicity, and immune evasion. HVT lacks homologues of MDV1 oncoprotein MEQ, CxC chemokine, oncogenicity-associated phosphoprotein pp24, and conserved domains of phosphoprotein pp38. These significant genomic differences in and adjacent to RS and RL regions likely account for the differences in host range, virulence, and oncogenicity between nonpathogenic HVT and highly pathogenic MDV1.
A Novel CD8-Independent High-Avidity Cytotoxic T-Lymphocyte Response Directed against an Epitope in the Phosphoprotein of the Paramyxovirus Simian Virus 5
Authors: Gray Peter M. , Parks Griffith D. , Alexander-Miller Martha A.
Published: July 2002

Adoptive transfer studies have shown that cytotoxic T lymphocytes (CTL) of high avidity, capable of recognizing low levels of peptide-MHC I molecules, are more efficient at reducing viral titers than are low-avidity CTL, thus establishing CTL avidity as a critical parameter for the ability of a CTL to clear virus in vivo. It has been well documented that CTL of high avidity are relatively CD8 independent, whereas low-avidity CTL require CD8 engagement in order to become activated. In this study we have analyzed the antiviral CTL response elicited following infection with the paramyxovirus simian virus 5 (SV5). We have identified the immunodominant and subdominant CTL responses and subsequently assessed the avidity of these responses by their CD8 dependence. This is the first study in which the relationship between immunodominance and CTL avidity has been investigated. The immunodominant response was directed against an epitope present in the viral M protein, and subdominant responses were directed against epitopes present in the P, F, and HN proteins. Similarly to other CTL responses we have analyzed, the immunodominant response and the subdominant F and HN responses were comprised of both high- and low-avidity CTL. However, the subdominant response directed against the epitope present in the P protein is novel, as it is exclusively high avidity. This high-avidity response is independent of both the route of infection and expression by recombinant SV5. A further understanding of the inherent properties of P that elicit only high-avidity CTL may allow for the design of more efficacious vaccine vectors that preferentially elicit high-avidity CTL in vivo.
CD4+T Cells Induced by a DNA Vaccine: Immunological Consequences of Epitope-Specific Lysosomal Targeting
Authors: Rodriguez Fernando , Harkins Stephanie , Redwine Jeffrey M. , de Pereda Jose M. , Whitton J. Lindsay
Published: July 2002

Our previous studies have shown that targeting DNA vaccine-encoded major histocompatibility complex class I epitopes to the proteasome enhanced CD8+T-cell induction and protection against lymphocytic choriomeningitis virus (LCMV) challenge. Here, we expand these studies to evaluate CD4+T-cell responses induced by DNA immunization and describe a system for targeting proteins and minigenes to lysosomes. Full-length proteins can be targeted to the lysosomal compartment by covalent attachment to the 20-amino-acid C-terminal tail of lysosomal integral membrane protein-II (LIMP-II). Using minigenes encoding defined T-helper epitopes from lymphocytic choriomeningitis virus, we show that the CD4+T-cell response induced by the NP309–328epitope of LCMV was greatly enhanced by addition of the LIMP-II tail. However, the immunological consequence of lysosomal targeting is not invariably positive; the CD4+T-cell response induced by the GP61–80epitope was almost abolished when attached to the LIMP-II tail. We identify the mechanism which underlies this marked difference in outcome. The GP61–80epitope is highly susceptible to cleavage by cathepsin D, an aspartic endopeptidase found almost exclusively in lysosomes. We show, using mass spectrometry, that the GP61–80peptide is cleaved between residues F74and K75and that this destroys its ability to stimulate virus-specific CD4+T cells. Thus, the immunological result of lysosomal targeting varies, depending upon the primary sequence of the encoded antigen. We analyze the effects of CD4+T-cell priming on the virus-specific antibody and CD8+T-cell responses which are mounted after virus infection and show that neither response appears to be accelerated or enhanced. Finally, we evaluate the protective benefits of CD4+T-cell vaccination in the LCMV model system; in contrast to DNA vaccine-induced CD8+T cells, which can confer solid protection against LCMV challenge, DNA vaccine-mediated priming of CD4+T cells does not appear to enhance the vaccinee's ability to combat viral challenge.
Matrix Protein and Another Viral Component Contribute to Induction of Apoptosis in Cells Infected with Vesicular Stomatitis Virus
Authors: Kopecky Sarah A. , Willingham Mark C. , Lyles Douglas S.
Published: July 2002

The induction of apoptosis in host cells is a prominent cytopathic effect of vesicular stomatitis virus (VSV) infection. The viral matrix (M) protein is responsible for several important cytopathic effects, including the inhibition of host gene expression and the induction of cell rounding in VSV-infected cells. This raises the question of whether M protein is also involved in the induction of apoptosis. HeLa or BHK cells were transfected with M mRNA to determine whether M protein induces apoptosis when expressed in the absence of other viral components. Expression of M protein induced apoptotic morphological changes and activated caspase-3 in both cell types, indicating that M protein induces apoptosis in the absence of other viral components. An M protein containing a point mutation that renders it defective in the inhibition of host gene expression (M51R mutation) activated little, if any, caspase-3, while a deletion mutant lacking amino acids 4 to 21 that is defective in the virus assembly function but fully functional in the inhibition of host gene expression was as effective as wild-type (wt) M protein in activating caspase-3. To determine whether M protein influences the induction of apoptosis in the context of a virus infection, the M51R M protein mutation was incorporated onto a wt background by using a recombinant infectious cDNA clone (rM51R-M virus). The timing of the induction of apoptosis by rM51R-M virus was compared to that by the corresponding recombinant wt (rwt) virus and to that by
ts
O82 virus, the mutant virus in which the M51R mutation was originally identified. In HeLa cells, rwt virus induced apoptosis faster than did rM51R-M virus, demonstrating a role for M protein in the induction of apoptosis. In contrast to the results obtained with HeLa cells, rwt virus induced apoptosis more slowly than did rM51R-M virus in BHK cells. This indicates that a viral component other than M protein contributes to induction of apoptosis in BHK cells and that wt M protein acts to delay induction of apoptosis by the other viral component.
ts
O82 virus induced apoptosis more rapidly than did rM51R-M virus in both HeLa and BHK cells. These two viruses contain the same point mutation in their M proteins, suggesting that sequence differences in genes other than that for M protein affect their rates of induction of apoptosis.
Characterization of the Structural Gene Promoter of
Aedes aegypti
Densovirus
Authors: Ward Todd W. , Kimmick Michael W. , Afanasiev Boris N. , Carlson Jonathan O.
Published: July 2002

Aedes aegypti
densonucleosis virus (AeDNV) has two promoters that have been shown to be active by reporter gene expression analysis (B. N. Afanasiev, Y. V. Koslov, J. O. Carlson, and B. J. Beaty, Exp. Parasitol. 79:322–339, 1994). Northern blot analysis of cells infected with AeDNV revealed two transcripts 1,200 and 3,500 nucleotides in length that are assumed to express the structural protein (VP) gene and nonstructural protein genes, respectively. Primer extension was used to map the transcriptional start site of the structural protein gene. Surprisingly, the structural protein gene transcript began at an initiator consensus sequence, CAGT, 60 nucleotides upstream from the map unit 61 TATAA sequence previously thought to define the promoter. Constructs with the β-galactosidase gene fused to the structural protein gene were used to determine elements necessary for promoter function. Deletion or mutation of the initiator sequence, CAGT, reduced protein expression by 93%, whereas mutation of the TATAA sequence at map unit 61 had little effect. An additional open reading frame was observed upstream of the structural protein gene that can express β-galactosidase at a low level (20% of that of VP fusions). Expression of the AeDNV structural protein gene was shown to be stimulated by the major nonstructural protein NS1 (Afanasiev et al., Exp. parasitol., 1994). To determine the sequences required for transactivation, expression of structural protein gene–β-galactosidase gene fusion constructs differing in AeDNV genome content was measured with and without NS1. The presence of NS1 led to an 8- to 10-fold increase in expression when either genomic end was present, compared to a 2-fold increase with a construct lacking the genomic ends. An even higher (37-fold) increase in expression occurred with both genomic ends present; however, this was in part due to template replication as shown by Southern blot analysis. These data indicate the location and importance of various elements necessary for efficient protein expression and transactivation from the structural protein gene promoter of AeDNV.
Murine Cytomegalovirus Open Reading Frame M27 Plays an Important Role in Growth and Virulence in Mice
Authors: Abenes Gerardo , Lee Manfred , Haghjoo Erik , Tong Tuong , Zhan Xiaoyan , Liu Fenyong
Published: July 2002

Using a Tn
3
-based transposon mutagenesis approach, we have generated a pool of murine cytomegalovirus (MCMV) mutants. In this study, one of the mutants, RvM27, which contained the transposon sequence at open reading frame M27, was characterized both in tissue culture and in immunocompetent BALB/c mice and immunodeficient SCID mice. Our results suggest that the M27 carboxyl-terminal sequence is dispensable for viral replication in vitro. Compared to the wild-type strain and a rescued virus that restored the M27 region, RvM27 was attenuated in growth in both BALB/c and SCID mice that were intraperitoneally infected with the viruses. Specifically, the titers of RvM27 in the salivary glands, lungs, spleens, livers, and kidneys of the infected SCID mice at 21 days postinfection were 50- to 500-fold lower than those of the wild-type virus and the rescued virus. Moreover, the virulence of the mutant virus appeared to be attenuated, because no deaths occurred among SCID mice infected with RvM27 for up to 37 days postinfection, while all the animals infected with the wild-type and rescued viruses died within 27 days postinfection. Our observations provide the first direct evidence to suggest that a disruption of M27 expression results in reduced viral growth and attenuated viral virulence in vivo in infected animals. Moreover, these results suggest that M27 is a viral determinant required for optimal MCMV growth and virulence in vivo and provide insight into the functions of the M27 homologues found in other animal and human CMVs as well as in other betaherpesviruses.
Type B Leukemogenic Virus Has a T-Cell-Specific Enhancer That Binds AML-1
Authors: Mertz Jennifer A. , Mustafa Farah , Meyers Shari , Dudley Jaquelin P.
Published: July 2002

Type B leukemogenic virus (TBLV) induces rapidly appearing T-cell tumors in mice. TBLV is highly related to mouse mammary tumor virus (MMTV) except that TBLV long terminal repeats (LTRs) have a deletion of negative regulatory elements and a triplication of sequences flanking the deletion. To determine if the LTR triplication represents a viral enhancer element, we inserted the triplication upstream and downstream in either orientation relative to the thymidine kinase promoter linked to the luciferase gene. These experiments showed that upregulation of reporter gene activity by the TBLV triplication was relatively orientation independent, consistent with the activity of eukaryotic enhancer elements. TBLV enhancer activity was observed in T-cell lines but not in fibroblasts, B cells, or mammary cells, suggesting that enhancer function is cell type dependent. To analyze the transcription factor binding sites that are important for TBLV enhancer function, we prepared substitution mutations in a reconstituted C3H MMTV LTR that recapitulates the deletion observed in the TBLV LTR. Transient transfections showed that a single mutation (556M) decreased TBLV enhancer activity at least 20-fold in two different T-cell lines. This mutation greatly diminished AML-1 (recently renamed RUNX1) binding in gel shift assays with a mutant oligonucleotide, whereas AML-1 binding to a wild-type TBLV oligomer was specific, as judged by competition and supershift experiments. The 556 mutation also reduced TBLV enhancer binding of two other protein complexes, called NF-A and NF-B, that did not appear to be related to c-Myb or Ets.AML-1overexpression in a mammary cell line enhanced expression from the TBLV LTR approximately 30-fold. These data suggest that binding of AML-1 to the TBLV enhancer, likely in combination with other factors, is necessary for optimal enhancer function.
Perinatal Transmission of Major, Minor, and Multiple Maternal Human Immunodeficiency Virus Type 1 Variants In Utero and Intrapartum
Authors: Dickover Ruth E. , Garratty Eileen M. , Plaeger Susan , Bryson Yvonne J.
Published: July 2002

Previous studies have provided conflicting data on the presence of selective pressures in the transmission of a homogeneous maternal viral subpopulation to the infant. Therefore, the purpose of this study was to definitively characterize the human immunodeficiency virus type 1 (HIV-1) quasispecies transmitted in utero and intrapartum. HIV-1 envelope gene diversity from peripheral blood mononuclear cells and plasma was measured during gestation and at delivery in mothers who did and did not transmit HIV perinatally by using a DNA heteroduplex mobility assay. Children were defined as infected in utero or intrapartum based on the timing of the first detection of HIV. Untreated transmitting mothers (
n
= 19) had significantly lower HIV-1 quasispecies diversity at delivery than untreated nontransmittting mothers (
n
= 18) (median Shannon entropy, 0.711 [0.642 to 0.816] versus 0.853 [0.762 to 0.925],
P
= 0.005). Eight mothers transmitted a single major
env
variant to their infants in utero, and one mother transmitted a single major
env
variant intrapartum. Four mothers transmitted multiple HIV-1
env
variants to their infants in utero, and two mothers transmitted multiple
env
variants intrapartum. The remaining six intrapartum- and two in utero-infected infants had a homogeneous HIV-1
env
quasispecies which did not comigrate with their mothers' bands at their first positive time point. In conclusion, in utero transmitters were more likely to transmit single or multiple major maternal viral variants. In contrast, intrapartum transmitters were more likely to transmit minor HIV-1 variants. These data indicate that different selective pressures, depending on the timing of transmission, may be involved in determining the pattern of maternal HIV-1 variant transmission.
Effect on Polyomavirus T-Antigen Function of Mutations in a Conserved Leucine-Rich Segment of the DnaJ Domain
Authors: Li Hongyun , Söderbärg Karin , Houshmand Hamid , You Zhi-Yong , Magnusson Göran
Published: July 2002

The N-terminal part of the mouse polyomavirus T antigens contains a highly conserved segment (-LLELLKL-), including amino acid residues 13 to 19. The sequence motif is predicted to form alpha helix I in the DnaJ domain of the T antigens. Four mutants with conservative substitutions of amino acid residues 13 and 14 were constructed. Of the four substitutions, L13M, L13I, L13V, and L14V, only L13V resulted in a phenotypic change. In transfected mouse cells, L13V large T antigen showed a more than 100-fold-reduced viral DNA synthesis. The viral replication could not be rescued by cotransfection of the cells with DNA expressing small t antigen or a large T antigen truncated at the C terminus that would compensate for a defect in host cell stimulation. In contrast to the effect on DNA replication, the L13V substitution in large T antigen did not prevent complex formation with Hsc70 and the Rb protein. Also, the activity of the protein in transactivation of transcription from the adenovirus E2 promoter was unimpaired, showing that the transcription factor E2F was released from pRb. The L13V substitution also caused a defect in small t antigen. However, this phenotypic change was due to protein instability. In contrast, middle T antigen with the L13V substitution remained stable and functional in cellular transformation. Together, the data show that the effect of the L13V substitution did not abrogate the Hsc70 interaction of the DnaJ domain. However, it is possible that the substitution of amino acid residue 13 affected specific DnaJ functions of large T antigen.
African Swine Fever Virus IAP Homologue Inhibits Caspase Activation and Promotes Cell Survival in Mammalian Cells
Authors: Nogal Marı́a L. , González de Buitrago Gonzalo , Rodrı́guez Clara , Cubelos Beatriz , Carrascosa Angel L. , Salas Marı́a L. , Revilla Yolanda
Published: July 2002

African swine fever virus (ASFV) A224L is a member of the inhibitor of apoptosis protein (IAP) family. We have investigated the antiapoptotic function of the viral IAP both in stably transfected cells and in ASFV-infected cells. A224L was able to substantially inhibit caspase activity and cell death induced by treatment with tumor necrosis factor alpha and cycloheximide or staurosporine when overexpressed in Vero cells by gene transfection. We have also observed that ASFV infection induces caspase activation and apoptosis in Vero cells. Furthermore, using a deletion mutant of ASFV lacking the A224L gene, we have shown that the viral IAP modulates the proteolytic processing of the effector cell death protease caspase-3 and the apoptosis which are induced in the infected cells. Our findings indicate that A224L interacts with the proteolytic fragment of caspase-3 and inhibits the activity of this protease during ASFV infection. These observations could indicate a conserved mechanism of action for ASFV IAP and other IAP family members to suppress apoptosis.
Activation of NFAT-Dependent Gene Expression by Nef: Conservation among Divergent Nef Alleles, Dependence on SH3 Binding and Membrane Association, and Cooperation with Protein Kinase C-θ
Authors: Manninen Aki , Huotari Päivi , Hiipakka Marita , Renkema G. Herma , Saksela Kalle
Published: July 2002

Here we show that the potential to regulate NFAT is a conserved property of different Nef alleles and that Nef residues involved in membrane targeting and SH3 binding are critical for this function. Cotransfection of an activated protein kinase C-θ (PKC-θ) with Nef implicated PKC-θ as a possible physiological cofactor of Nef in promoting NFAT-dependent gene expression and T-cell activation.
Membrane-Anchored Peptide Inhibits Human Immunodeficiency Virus Entry
Authors: Hildinger Markus , Dittmar Matthias T. , Schult-Dietrich Patricia , Fehse Boris , Schnierle Barbara S. , Thaler Sonja , Stiegler Gabriela , Welker Reinhold , von Laer Dorothee
Published: July 2002

Peptides derived from the heptad repeats of human immunodeficiency virus (HIV) gp41 envelope glycoprotein, such as T20, can efficiently inhibit HIV type 1 (HIV-1) entry. In this study, replication of HIV-1 was inhibited more than 100-fold in a T-helper cell line transduced with a retrovirus vector expressing membrane-anchored T20 on the cell surface. Inhibition was independent of coreceptor usage.
Infection of the CD45RA+(Naive) Subset of Peripheral CD8+Lymphocytes by Human Immunodeficiency Virus Type 1 In Vivo
Authors: McBreen S. , Imlach S. , Shirafuji T. , Scott G. R. , Leen C. , Bell J. E. , Simmonds P.
Published: July 2002

To investigate the mechanism and functional significance of infection of CD8+lymphocytes by human immunodeficiency virus type 1 (HIV-1) in vivo, we determined frequencies of infection, proviral conformation, and genetic relationships between HIV-1 variants infecting naive (CD45RA+) and memory (CD45RO+) peripheral blood CD4+and CD8+lymphocytes. Infection of CD3+CD8+CD45RA+cells was detected in 9 of 16 study subjects at frequencies ranging from 30 to 1,400 proviral copies/106cells, more frequently than CD3+CD8+lymphocytes expressing the RO isoform of CD45 (n= 2, 70 and 260 copies /106cells). In agreement with previous studies, there was no evidence for a similar preferential infection of CD4+naive lymphocytes. Proviral sequences in both CD4+and CD8+lymphocyte subsets were complete, as assessed by quantitation using primers from the long terminal repeat region spanning the tRNA primer binding site. In six of the seven study subjects investigated, variants infecting CD8+lymphocytes were partially or completely genetically distinct in the V3 region from those recovered from CD4+lymphocytes and showed a greater degree of compartmentalization than observed between naive and memory subsets of CD4+lymphocytes. In two study subjects, arginine substitutions at position 306, associated with use of the chemokine coreceptor CXCR4, were preferentially found in CD4 lymphocytes. These population differences may have originated through different times of infection rather than necessarily indicating a difference in their biological properties. The preferential distribution of HIV-1 in naive CD8+lymphocytes indeed suggests that infection occurred early in T-lymphocyte ontogeny, such as during maturation in the thymus. Destruction of cells destined to become CD8+lymphocytes may be a major factor in the decline in CD8+lymphocyte frequencies and function on disease progression and may contribute directly to the observed immunodeficiency in AIDS.
Two Antigenic Peptides from Genes
m123
and
m164
of Murine Cytomegalovirus Quantitatively Dominate CD8 T-Cell Memory in the
H-2

d

Haplotype
Authors: Holtappels Rafaela , Thomas Doris , Podlech Jürgen , Reddehase Matthias J.
Published: July 2002

The importance of CD8 T cells for the control of cytomegalovirus (CMV) infection has raised interest in the identification of immunogenic viral proteins as candidates for vaccination and cytoimmunotherapy. The final aim is to determine the viral “immunome” for any major histocompatibility complex class I molecule by antigenicity screening of proteome-derived peptides. For human CMV, there is a limitation to this approach: the T cells used as responder cells for peptide screening are usually memory cells that have undergone in vivo selection. On this basis, pUL83 (pp65) and pUL123 (IE1 or pp68 to -72) were classified as immunodominant proteins. It is an open question whether this limited “memory immunome” really reflects the immunogenic potential of the human CMV proteome. Here we document an analogous focus of the memory repertoire on two proteins of murine CMV. Specifically, ca. 80% of all memory CD8 T cells in the spleen as well as in persisting pulmonary infiltrates were found to be specific for the known IE1 peptide
168
YPHFMPTNL
176
and for the peptide
257
AGPPRYSRI
265
, newly defined here, derived from open reading frame
m164
. Notably, CD8 T-cell lines of both specificities protected against acute infection upon adoptive transfer. In contrast, the natural immune response to acute infection in draining lymph nodes and in the lungs indicated a somewhat broader specificity repertoire. We conclude that the low number of antigenic peptides identified so far for CMVs reflects a focused memory repertoire, and we predict that more antigenic peptides will be disclosed by analysis of the acute immune response.
Activation of NF-κB by R5 and X4 Human Immunodeficiency Virus Type 1 Induces Macrophage Inflammatory Protein 1α and Tumor Necrosis Factor Alpha in Macrophages
Authors: Choe Wonkyu , Volsky David J. , Potash Mary Jane
Published: July 2002

NF-κB is a transcriptional activator that often regulates inflammatory responses. We demonstrate that human immunodeficiency virus type 1 activates nuclear localization of NF-κB in macrophages in a manner dependent upon virus strain but independent of virus replication. Through the use of an inhibitor, NF-κB activation was found to be responsible for the cytokine and chemokine induction that we recently reported.
Sequencing of Porcine Enterovirus Groups II and III Reveals Unique Features of Both Virus Groups
Authors: Krumbholz Andi , Dauber Malte , Henke Andreas , Birch-Hirschfeld Eckhard , Knowles Nick J. , Stelzner Axel , Zell Roland
Published: July 2002

The molecular classification of the porcine enterovirus (PEV) groups II and III was investigated. The sequence of the almost complete PEV-8 (group II) genome reveals that this virus has unique L and 2A gene regions. A reclassification of this group into a new picornavirus genus is suggested. PEV group III viruses are typical enteroviruses. They differ from other enteroviruses by a prolonged stem-loop D of the 5′-cloverleaf structure.
Detection of Viral DNA and Immune Responses to the Human Herpesvirus 6 101-Kilodalton Virion Protein in Patients with Multiple Sclerosis and in Controls
Authors: Tejada-Simon Maria V. , Zang Ying C. Q. , Hong Jian , Rivera Victor M. , Killian James M. , Zhang Jingwu Z.
Published: July 2002

Human herpesvirus 6 (HHV-6), a latent lymphotropic and neurotropic virus, has been suspected as an etiologic agent in multiple sclerosis (MS). The study was undertaken to correlate virologic evidence for HHV-6 activity with the state of host immunity to HHV-6 in MS patients and control subjects. The study revealed that cell-free DNA of HHV-6 was detected more frequently in both serum and cerebrospinal fluid of MS patients than in those of control subjects. T cells recognizing the recombinant 101-kDa protein (101K) corresponding to the major immunoreactive region unique to HHV-6 occurred at significantly lower precursor frequency in MS patients than in control subjects. The resulting HHV-6-specific T-cell lines obtained from MS patients exhibited skewed cytokine profiles characterized by the inability to produce interleukin-4 (IL-4) and IL-10. The decreased T-cell responses to HHV-6 and the altered cytokine profile were consistent with significantly declined serum immunoglobulin G (IgG) titers for HHV-6 of MS patients compared to those of control subjects. In contrast, elevated serum IgM titers for HHV-6 were detected in the majority of MS patients, which may reflect frequent exposure of B cells to HHV-6. The findings suggest that the decreased immune responses to HHV-6 may be responsible for ineffective clearance of HHV-6 in MS patients.
The p23 Protein of
Citrus Tristeza Virus
Controls Asymmetrical RNA Accumulation
Authors: Satyanarayana Tatineni , Gowda Siddarame , Ayllón María A. , Albiach-Martí María R. , Rabindran Shailaja , Dawson William O.
Published: July 2002

Citrus tristeza virus
(CTV), a member of the
Closteroviridae
, has a 19.3-kb positive-stranded RNA genome that is organized into 12 open reading frames (ORFs) with the 10 3′ genes expressed via a nested set of nine or ten 3′-coterminal subgenomic mRNAs (sgRNAs). Relatively large amounts of negative-stranded RNAs complementary to both genomic and sgRNAs accumulate in infected cells. As is characteristic of RNA viruses, wild-type CTV produced more positive than negative strands, with the plus-to-minus ratios of genomic and sgRNAs estimated at 10 to 20:1 and 40 to 50:1, respectively. However, a mutant with all of the 3′ genes deleted replicated efficiently, but produced plus to minus strands at a markedly decreased ratio of 1 to 2:1. Deletion analysis of 3′-end genes revealed that the p23 ORF was involved in asymmetric RNA accumulation. A mutation which caused a frameshift after the fifth codon resulted in nearly symmetrical RNA accumulation, suggesting that the p23 protein, not a
cis
-acting element within the p23 ORF, controls asymmetric accumulation of CTV RNAs. Further in-frame deletion mutations in the p23 ORF suggested that amino acid residues 46 to 180, which contained RNA-binding and zinc finger domains, were indispensable for asymmetrical RNA accumulation, while the N-terminal 5 to 45 and C-terminal 181 to 209 amino acid residues were not absolutely required. Mutation of conserved cysteine residues to alanines in the zinc finger domain resulted in loss of activity of the p23 protein, suggesting involvement of the zinc finger in asymmetric RNA accumulation. The absence of p23 gene function was manifested by substantial increases in accumulation of negative-stranded RNAs and only modest decreases in positive-stranded RNAs. Moreover, the substantial decrease in the accumulation of negative-stranded coat protein (CP) sgRNA in the presence of the functional p23 gene resulted in a 12- to 15-fold increase in the expression of the CP gene. Apparently the excess negative-stranded sgRNA reduces the availability of the corresponding positive-stranded sgRNA as a messenger. Thus, the p23 protein controls asymmetric accumulation of CTV RNAs by downregulating negative-stranded RNA accumulation and indirectly increases expression of 3′ genes.
Characterization of E3/49K, a Novel, Highly Glycosylated E3 Protein of the Epidemic Keratoconjunctivitis-Causing Adenovirus Type 19a
Authors: Windheim Mark , Burgert Hans-Gerhard
Published: July 2002

The early transcription unit 3 (E3) of human adenoviruses (Ads) encodes proteins with various immunomodulatory functions. Ads from different subgenera differ considerably in their E3 coding capacity, suggesting that distinct sets of immunomodulatory E3 proteins may influence the disease pattern associated with different Ad subgenera. Interestingly, the E3 region of Ads classified in subgenus D, which are often isolated from AIDS patients and have the propensity to cause eye infections, contains a unique gene, named
E3/49K
, that may encode a protein with a calculated molecular weight of 48,984 that might be implicated in diseases caused by this subgenus. The 49K sequence predicts a highly glycosylated type I transmembrane protein with a short cytoplasmic tail containing two motifs, YXXΦ and LL, potentially involved in targeting the protein to endosomal or lysosomal compartments. Remarkably, the 49K protein is predicted to contain an unusual immunoglobulin-like fold. Here we have characterized the E3/49K protein of Ad type 19a, an Ad of subgenus D which causes epidemic keratoconjunctivitis. E3/49K was synthesized as an 80- to 100-kDa protein, which is unusually large for an E3 protein. In contrast to another early protein, E3/19K, the expression of E3/49K started early but continued throughout the infection cycle. Analysis of the 49K glycosylation revealed that the majority of 49K molecules contained only 12 of the predicted 14 N-glycans. Furthermore, we provide evidence that 49K is O-glycosylated. At steady state, E3/49K was localized in the Golgi-
trans
-Golgi network and in early endosomes. Interestingly, the 49K protein has a rather short half-life and seems to be proteolytically cleaved. A processing pattern similar to that in the early stages of infection is seen in transfected cells, constitutively expressing 49K in the absence of other Ad proteins. Together, our data provide the first biochemical and cell biological characterization of an unique E3 protein of subgenus D Ads.
Efficient and Selective Gene Transfer into Primary Human Brain Tumors by Using Single-Chain Antibody-Targeted Adenoviral Vectors with Native Tropism Abolished
Authors: van Beusechem Victor W. , Grill Jacques , Mastenbroek D. C. Jeroen , Wickham Thomas J. , Roelvink Peter W. , Haisma Hidde J. , Lamfers Martine L. M. , Dirven Clemens M. F. , Pinedo Herbert M. , Gerritsen Winald R.
Published: July 2002

The application of adenoviral vectors in cancer gene therapy is hampered by low receptor expression on tumor cells and high receptor expression on normal epithelial cells. Targeting adenoviral vectors toward tumor cells may improve cancer gene therapy procedures by providing augmented tumor transduction and decreased toxicity to normal tissues. Targeting requires both the complete abolition of native tropism and the addition of a new specific binding ligand onto the viral capsid. Here we accomplished this by using doubly ablated adenoviral vectors, lacking coxsackievirus-adenovirus receptor and α
v
integrin binding capacities, together with bispecific single-chain antibodies targeted toward human epidermal growth factor receptor (EGFR) or the epithelial cell adhesion molecule. These vectors efficiently and selectively targeted both alternative receptors on the surface of human cancer cells. Targeted doubly ablated adenoviral vectors were also very efficient and specific with primary human tumor specimens. With primary glioma cell cultures, EGFR targeting augmented the median gene transfer efficiency of doubly ablated adenoviral vectors 123-fold. Moreover, EGFR-targeted doubly ablated vectors were selective for human brain tumors versus the surrounding normal brain tissue. They transduced organotypic glioma and meningioma spheroids with efficiencies similar to those of native adenoviral vectors, while exhibiting greater-than-10-fold-reduced background levels on normal brain explants from the same patients. As a result, EGFR-targeted doubly ablated adenoviral vectors had a 5- to 38-fold-improved tumor-to-normal brain targeting index compared to native vectors. Hence, single-chain targeted doubly ablated adenoviral vectors are promising tools for cancer gene therapy. They should provide an improved therapeutic index with efficient tumor transduction and effective protection of normal tissue.
Inhibition of Human Immunodeficiency Virus Type 1 Activity In Vitro by a New Self-Stabilized Oligonucleotide with Guanosine-Thymidine Quadruplex Motifs
Authors: Suzuki Jun-ichiro , Miyano-Kurosaki Naoko , Kuwasaki Tomoyuki , Takeuchi Hiroaki , Kawai Gota , Takaku Hiroshi
Published: July 2002

An oligonucleotide with a dimeric hairpin guanosine quadruplex (basket type structure) (dG3T4G3-s), containing phosphorothioate groups, was able to inhibit human immunodeficiency virus type 1 (HIV-1)-induced syncytium formation and virus production (as measured by p24 core antigen expression) in peripheral blood mononuclear cells. This oligonucleotide lacks primary sequence homology with the complementary (antisense) sequences to the HIV-1 genome. Furthermore, this oligonucleotide may have increased nuclease resistance. The activity of this oligonucleotide was increased when the phosphodiester backbone was replaced with a phosphorothioate backbone. In vivo results showed that dG3T4G3-s was capable of blocking the interaction between gp120 and CD4. We also found that dG3T4G3-s specifically inhibits the entry of T-cell line-tropic HIV-1 into cells. This compound is a viable candidate for evaluation as a therapeutic agent against HIV-1 in humans.
Increased RNA Editing and Inhibition of Hepatitis Delta Virus Replication by High-Level Expression of ADAR1 and ADAR2
Authors: Jayan Geetha C. , Casey John L.
Published: July 2002

Hepatitis delta virus (HDV) is a subviral human pathogen that uses specific RNA editing activity of the host to produce two essential forms of the sole viral protein, hepatitis delta antigen (HDAg). Editing at the amber/W site of HDV antigenomic RNA leads to the production of the longer form (HDAg-L), which is required for RNA packaging but which is a potent
trans
-dominant inhibitor of HDV RNA replication. Editing in infected cells is thought to be catalyzed by one or more of the cellular enzymes known as adenosine deaminases that act on RNA (ADARs). We examined the effects of increased ADAR1 and ADAR2 expression on HDV RNA editing and replication in transfected Huh7 cells. We found that both ADARs dramatically increased RNA editing, which was correlated with strong inhibition of HDV RNA replication. While increased HDAg-L production was the primary mechanism of inhibition, we observed at least two additional means by which ADARs can suppress HDV replication. High-level expression of both ADAR1 and ADAR2 led to extensive hyperediting at non-amber/W sites and subsequent production of HDAg variants that acted as
trans
-dominant inhibitors of HDV RNA replication. Moreover, we also observed weak inhibition of HDV RNA replication by mutated forms of ADARs defective for deaminase activity. Our results indicate that HDV requires highly regulated and selective editing and that the level of ADAR expression can play an important role: overexpression of ADARs inhibits HDV RNA replication and compromises virus viability.
Fatty Acids on the A/USSR/77 Influenza Virus Hemagglutinin Facilitate the Transition from Hemifusion to Fusion Pore Formation
Authors: Sakai Tatsuya , Ohuchi Reiko , Ohuchi Masanobu
Published: July 2002

Influenza virus hemagglutinin (HA) has three highly conserved acylation sites close to the carboxyl terminus of the HA2 subunit, one in the transmembrane domain and two in the cytoplasmic domain. Each site is modified by palmitic acid through a thioester linkage to cysteine. To elucidate the biological significance of HA acylation, the acylation sites of HA of influenza virus strain A/USSR/77 (H1N1) were changed by site-directed mutagenesis, and the membrane fusion activity of mutant HAs lacking the acylation site(s) was examined quantitatively using transfer assays of lipid (R18) and aqueous (calcein) dyes. Lipid mixing, so-called hemifusion, activity was not affected by deacylation, whereas transfer of aqueous dye, so-called fusion pore formation, was dramatically restricted. When the fusion reaction was induced by a lower pH than the optimal one, calcein transfer with the mutant HAs was improved, but simultaneously a considerable calcein leakage into the medium was observed. From these results, we conclude that the palmitic acids on the H1 subtype HA facilitate the transition from hemifusion to fusion pore formation.
Influence of Particle Shape on the Drag Coefficient for Isometric Particles
Authors: Hartman Miloslav , Trnka Otakar , Svoboda Karel , Veselý Václav
Published: July 2002

A comprehensive correlation has been developed of the drag coefficient for nonspherical isometric particles as a function the Reynolds number and the particle sphericity on the basis of data reported in the literature. The proposed formula covers the Stokes, the transitional and the Newton region. The predictions of the reported correlation have been compared to experimental data measured in this work with the dolomitic materials in respect to their use in calcination and gas cleaning processes with fluidized beds. Approximative explicit formulae have also been reported that make it possible to estimate the terminal free-fall velocity of a given particle or to predict the particle diameter corresponding to a fluid velocity of interest.
Polyaniline: Forms and Formation
Authors: Stejskal Jaroslav , Kratochvíl Pavel , Jenkins Aubrey D.
Published: July 2002

The forms of polyaniline, viz. pernigraniline, emeraldine and leucoemeraldine, their mutual transformations and their roles in the chemical polymerization of aniline, are discussed. Absorption spectra of its various protonated forms and corresponding bases are reported. A simple reaction scheme is proposed to account for the colour and acidity changes during polymerization.
Synthesis of 2-Substituted Benzothiazoles Containing Amino Acid, Imino or Heteroaryl Moieties with Anticipated Fungicidal Activity
Authors: Nawwar Galal A. M. , Shafik Nancy A.
Published: July 2002

2-Aminothiophenol reacted with cyanoacetic acid derivatives to form 2-substituted benzothiazoles, which were used for further cyclization to yield quinoline, pyrazole or dihydropyridine derivatives.
Catalytic Effect of Active Carbon Black Chezacarb in Wet Oxidation of Phenol
Authors: Tukač Vratislav , Hanika Jiří
Published: July 2002

The catalytic effects of hydrophilic granulated active carbon black Chezacarb have been tested in the oxidation of dilute (5 000 ppm) aqueous solutions of phenol. The research was focused on combined utilization of sorption and chemical (oxygen functional groups) properties of carbon black which support formation of free radicals in oxidation and on the catalytic effects of metals (Fe, Ni and V) present in the sorbent mentioned. The oxidation was carried out in a stainless steel autoclave of 1.25 l capacity. The reaction temperatures and the total pressures varied in the intervals of 120-160 °C and 3-5 MPa, respectively. The reaction mixture was analyzed by means of HPLC with UV detection (254 nm). The experimental data were evaluated by means of the ASPEN PLUS 9.2 program. The model of laboratory autoclave involved the reaction kinetics as well as the phase equilibria and dissolution of oxygen and carbon dioxide in the reaction mixture. Although the active carbon black shows a lower catalytic activity than the usual copper catalyst in the wet oxidation, it has the advantage of avoiding the undesirable releases of heavy metals in waste waters, which escapes accompany the application of copper catalysts.
Synthesis of 2-Arylfuro[3,2-c]pyridines and Their Derivatives
Authors: Krutošíková Alžbeta , Sleziak Róbert
Published: July 2002

A series of 2-arylfuro[3,2-c]pyridines was synthesized. 3-(5-Aryl-2-furyl)propenoic acids 1a-1h were converted to the acid azides 2a-2h, which in turn were cyclized to give 2-arylfuro[3,2-c]pyridine-4(5H)-ones 4a-4h by heating in Dowtherm. The pyridones 4a-4f were aromatized with phosphorus oxychloride to the 2-aryl-4-chlorofuro[3,2-c]pyridines 5a-5f, which were reduced with zinc and acetic acid to the title compounds 6a-6f. Reacted with phosphorus(V) sulfide, the pyridones 4a-4f yielded the corresponding thiones 7a-7f.
Photoinitiated Degradation of 4-Chlorophenoxyacetic Acid by Hydrogen Peroxide; Photocatalytic Effects of Fe(III) Compounds
Authors: Luňák Stanislav , Brodilová Jiřina , Muzart Jacques
Published: July 2002

Photolysis of 4-chlorophenoxyacetic acid (4-CPA) and the effect of Fe(III) compounds on photochemical oxidative degradation of 4-CPA by hydrogen peroxide were studied. Whereas degradation initiated by 254 nm radiation is affected only moderately by the presence of Fe(III) compounds the reaction initiated by VIS radiation (high-pressure sodium lamp) is photocatalyzed and photosensitized by Fe(III) compounds very markedly. 4-Chlorophenol (4-CP) and phenol were detected as the main degradation intermediates. Formation of phenol prevails in the absence of oxidation agent, 4-CP is the main product in the presence of hydrogen peroxide.
Platinum, Palladium and Rhodium Clusters in Alkali X and Y Zeolites. Preparation and Catalytic Activity
Authors: Nováková Jana
Published: July 2002

Pt, Pd and Rh ammine cations partially ion-exchanged for alkali ions of X and Y zeolites were decomposed in vacuum to metallic clusters embedded in cavities. The course of the parent ammine decomposition as well as the CO + NO reaction after the deammination to metallic clusters were compared for the individual metals. The effects of the respective metals as well as as those of the zeolitic matrices are discussed.
On the Calculation of Transition State Activity Coefficient and Solvent Effects on Chemical Reactions
Authors: Domínguez Alvaro , Jimenez Rafael , López-Cornejo Pilar , Pérez Pilar , Sánchez Francisco
Published: July 2002

Solvent effects, when the classical transition state theory (TST) holds, can be interpreted following the Brønsted equation. However, when calculating the activity coefficient of the transition state, γ# it is important to take into account that this coefficient is different from that of the precursor complex, γPC. The activity coefficient of the latter is, in fact, that calculated in classical treatments of salt and solvent effects. In this paper it is shown how the quotients γ#/γPC change when the reaction medium changes. Therefore, the conclusions taken on the basis of classical treatments may be erroneous.
Bromination of Tetralin. Short and Efficient Synthesis of 1,4-Dibromonaphthalene
Authors: Çakmak Osman , Kahveci Ismail , Demirtaş Íbrahim , Hökelek Tuncer , Smith Keith
Published: July 2002

High-temperature bromination of tetralin (1,2,3,4-tetrahydronaphthalene) with bromine resulted in benzylic bromination to give 1,4-dibromo-1,2,3,4-tetrahydronaphthalene (4) as a major product and several secondary products. Photolytic bromination of tetralin and subsequent double dehydrobromination of 1,1,4,4-tetrabromo-1,2,3,4-tetrahydronaphthalene (10) gave 1,4-dibromonaphthalene (11) as the sole product in a high yield. 1,4-Dibromonaphthalene is efficiently converted to the corresponding methoxy (12 and 13) and cyano (14 and 15) derivatives of naphthalene.