bluuebunny/crossref_metadata_2025 huggingface dataset:
S
-Adenosylmethionine Decarboxylase from the Archaeon
Methanococcus jannaschii
: Identification of a Novel Family of Pyruvoyl Enzymes
Authors: Kim Alexander D. , Graham David E. , Seeholzer Steven H. , Markham George D.
Published: July 2002

Polyamines are present in high concentrations in archaea, yet little is known about their synthesis, except by extrapolation from bacterial and eucaryal systems.
S
-Adenosylmethionine (AdoMet) decarboxylase, a pyruvoyl group-containing enzyme that is required for spermidine biosynthesis, has been previously identified in eucarya and
Escherichia coli
. Despite spermidine concentrations in the
Methanococcales
that are several times higher than in
E. coli
, no AdoMet decarboxylase gene was recognized in the complete genome sequence of
Methanococcus jannaschii
. The gene encoding AdoMet decarboxylase in this archaeon is identified herein as a highly diverged homolog of the
E. coli speD
gene (less than 11% identity). The
M. jannaschii
enzyme has been expressed in
E. coli
and purified to homogeneity. Mass spectrometry showed that the enzyme is composed of two subunits of 61 and 63 residues that are derived from a common proenzyme; these proteins associate in an (αβ)
2
complex. The pyruvoyl-containing subunit is less than one-half the size of that in previously reported AdoMet decarboxylases, but the holoenzyme has enzymatic activity comparable to that of other AdoMet decarboxylases. The sequence of the
M. jannaschii
enzyme is a prototype of a class of AdoMet decarboxylases that includes homologs in other archaea and diverse bacteria. The broad phylogenetic distribution of this group suggests that the canonical SpeD-type decarboxylase was derived from an archaeal enzyme within the gamma proteobacterial lineage. Both SpeD-type and archaeal-type enzymes have diverged widely in sequence and size from analogous eucaryal enzymes.
Component of the
Rhodospirillum centenum
Photosensory Apparatus with Structural and Functional Similarity to Methyl-Accepting Chemotaxis Protein Chemoreceptors
Authors: Jiang Ze-Yu , Bauer Carl E.
Published: July 2002

Photosynthetic bacteria respond to alterations in light conditions by migrating to locations that allows optimal use of light as an energy source. Studies have indicated that photosynthesis-driven electron transport functions as an attractant signal for motility among purple photosynthetic bacteria. However, it is unclear just how the motility-based signal transduction system monitors electron flow through photosynthesis-driven electron transport. Recently, we have demonstrated that the purple photosynthetic bacterium
Rhodospirillum centenum
is capable of rapidly moving swarm cell colonies toward infrared light as well as away from visible light. Light-driven colony motility of
R. centenum
has allowed us to perform genetic dissection of the signaling pathway that affects photosynthesis-driven motility. In this study, we have undertaken sequence and mutational analyses of one of the components of a signal transduction pathway, Ptr, which appears responsible for transmitting a signal from the photosynthesis-driven electron transport chain to the chemotaxis signal transduction cascade. Mutational analysis demonstrates that cells disrupted for
ptr
are defective in altering motility in response to light, as well as defective in light-dependent release of methanol. We present a model which proposes that Ptr senses the redox state of a component in the photosynthetic cyclic electron transport chain and that Ptr is responsible for transmitting a signal to the chemotaxis machinery to induce a photosynthesis-dependent motility response.
Intragenic Suppressors of an OmpF Assembly Mutant and Assessment of the Roles of Various OmpF Residues in Assembly through Informational Suppressors
Authors: Kloser Andrew W. , Reading Jared T. , McDermott Tom , Stidham Rhesa , Misra Rajeev
Published: July 2002

We employed two separate genetic approaches to examine the roles of various OmpF residues in assembly. In one approach, intragenic suppressors of a temperature-sensitive OmpF assembly mutant carrying a W214E substitution were sought at 42°C, or at 37°C in a genetic background lacking the periplasmic folding factor SurA. In the majority of cases (58 out of 61 revertants), the suppressors mapped either at the original site (position 214) or two residues downstream from it. In the remaining three revertants that were obtained in a
surA
background, an alteration of N230Y was located 16 residues away from the original site. The N230Y suppressor also corrected OmpF315 assembly at 42°C in a
surA
+
background, indicating that the two different physiological environments imposed similar assembly constraints. The specificity of N230Y was tested against five different residues at position 214 of mature OmpF. Clear specificity was displayed, with maximum suppression observed for the original substitution at position 214 (E214) against which the N230Y suppressor was isolated, and no negative effect on OmpF assembly was noted when the wild-type W214 residue was present. The mechanism of suppression may involve compensation for a specific conformational defect. The second approach involved the application of informational suppressors (Su-tRNA) in combination with
ompF
amber mutations to generate variant OmpF proteins. In this approach we targeted the Y40, Q66, W214, and Y231 residues of mature OmpF and replaced them with S, Q, L, and Y through the action of Su-tRNAs. Thus, a total of 16 variant OmpF proteins were generated, of which three were identical to the parental protein, and two variants carrying W214Q and Y231Q substitutions were similar to assembly-defective proteins isolated previously (R. Misra, J. Bacteriol. 175:5049–5056, 1993). The results obtained from these analyses provided useful information regarding the compatibility of various alterations in OmpF assembly.
Identification of
yacE
(
coaE
) as the Structural Gene for Dephosphocoenzyme A Kinase in
Escherichia coli
K-12
Authors: Mishra Pranab k. , Park Peter K. , Drueckhammer Dale G.
Published: July 2002

Dephosphocoenzyme A (dephospho-CoA) kinase catalyzes the final step in coenzyme A biosynthesis, the phosphorylation of the 3′-hydroxy group of the ribose sugar moiety. Wild-type dephospho-CoA kinase from
Corynebacterium ammoniagenes
was purified to homogeneity and subjected to N-terminal sequence analysis. A BLAST search identified a gene from
Escherichia coli
previously designated
yacE
encoding a highly homologous protein. Amplification of the gene and overexpression yielded recombinant dephospho-CoA kinase as a 22.6-kDa monomer. Enzyme assay and nuclear magnetic resonance analyses of the product demonstrated that the recombinant enzyme is indeed dephospho-CoA kinase. The activities with adenosine, AMP, and adenosine phosphosulfate were 4 to 8% of the activity with dephospho-CoA. Homologues of the
E. coli
dephospho-CoA kinase were identified in a diverse range of organisms.
Characterization of Extended-Spectrum β-Lactamase (TEM-52)-Producing Strains of
Salmonella
enterica
Serovar Typhimurium with Diverse Resistance Phenotypes
Authors: Vahaboglu Haluk , Fuzi Miklos , Cetin Sila , Gundes Sibel , Ujhelyi Eniko , Coskunkan Figen , Tansel Ozlem
Published: July 2002

Two
Salmonella
enterica
serovar Typhimurium strains from different clonal origins, both producing an extended-spectrum β-lactamase (TEM-52), were isolated from a patient. This enzyme was encoded on a single plasmid and was found at very low levels in one strain, while being encoded on multiple plasmids and in multiple different
Eco
RI fragments in the other strain.
Evaluation of an Automated Liquid-Handling System (Tecan Genesis RSP 100) in the Abbott LCx Assay for
Chlamydia trachomatis
Authors: Hanson Kevan L. , Cartwright Charles P.
Published: July 2002

The present study investigated the feasibility of automating the specimen-pipetting component of sample preparation in the LCx Chlamydia assay (LCx-CT assay; Abbott Laboratories, Chicago, Ill.) by using a commercially available liquid-handling system (Tecan Genesis RSP100; Tecan Inc., Research Triangle Park, N.C.). The Tecan instrument proved to be comparable in both precision and accuracy to a manual multipipettor (Eppendorf model 4850; Eppendorf Scientific, Westbury, N.Y.). The Tecan instrument was extensively checked for evidence of specimen-to-specimen transfer, and no level of contamination sufficient to generate a signal above the background in the LCx-CT assay was detected. Finally, pipetting speed was significantly improved by using the Tecan instrument. A mean time of 2.5 min was required to pipette a complete LCx-CT assay carousel (20 samples and 4 controls) with the Tecan instrument, whereas 8.4 min was required to pipette a comparable number of samples manually (
P
< 0.001).
The Virus-Specific and Allospecific Cytotoxic T-Lymphocyte Response to Lymphocytic Choriomeningitis Virus Is Modified in a Subpopulation of CD8+T Cells Coexpressing the Inhibitory Major Histocompatibility Complex Class I Receptor Ly49G2
Authors: Peacock Craig D. , Lin Meei Y. , Ortaldo John R. , Welsh Raymond M.
Published: July 2002

The role of negatively signaling NK cell receptors of the Ly49 family on the specificity of the acute CD8+cytotoxic T-lymphocyte (CTL) response was investigated in lymphocytic choriomeningitis virus (LCMV)-infected C57BL/6 mice. Activated CD8+T cells coexpressing Ly49G2 expanded during LCMV infection, and T-cell receptor analyses by flow cytometry and CDR3 spectratyping revealed a unique polyclonal T-cell population in the Ly49G2+fraction. These cells lysed syngeneic targets infected with LCMV or coated with two of three LCMV immunodominant peptides examined. Transfection of these sensitive targets with H2Dd, a ligand for Ly49G2, inhibited lysis. This was reversed by antibody to Ly49G2, indicating effective negative signaling. LCMV characteristically induces an anti-H2dallospecific T-cell response that includes T-cell clones cross-reactive between allogeneic and LCMV-infected syngeneic targets. The CD8+Ly49G2+population mediated no allospecific killing, nor was any NK-like killing observed against YAC-1 cells. This study shows that CD8+Ly49G2+cells participate in the virus-induced CTL response but lyse a more restricted range of targets than the rest of the virus-induced CTL population.
Evaluation of VP22 Spread in Tissue Culture
Authors: Brewis Neil , Phelan Anne , Webb Jeanette , Drew Jeff , Elliott Gill , O'Hare Peter
Published: July 2002

We compare methods of detection of intercellular transport of the herpes simplex virus protein VP22 and of a green fluorescent protein (GFP)-VP22 fusion protein. Spread of both proteins was observed by immunofluorescence (IF) using organic fixatives. Spread of both proteins was also detected by IF after paraformaldehyde (PFA) fixation and detergent permeabilization, albeit at reduced levels. However, while spread of GFP-VP22 was observed by examining intrinsic GFP fluorescence after methanol fixation, little spread was observed after PFA fixation, suggesting that the levels of the fusion protein in recipient cells were below the detection limits of intrinsic-fluorescence or that PFA fixation quenches the fluorescence of GFP-VP22. We further considered whether elution of VP22 from methanol-fixed cells and postfixation binding to surrounding cells contributed to the increased detection of spread observed after methanol fixation. The results show that while this could occur, it appeared to be a minor effect not accounting for the observed VP22 cell-to-cell spread in culture.
Long Terminal Repeat Regions from Exogenous but Not Endogenous Feline Leukemia Viruses Transactivate Cellular Gene Expression
Authors: Ghosh Sajal K. , Roy-Burman Pradip , Faller Douglas V.
Published: July 2002

We have previously reported that the long terminal repeat (LTR) region of feline leukemia viruses (FeLVs) can enhance expression of certain cellular genes such as the collagenase IV gene andMCP-1intrans(S. K. Ghosh and D. V. Faller, J. Virol. 73:4931–4940, 1999). Genomic DNA of all healthy feline species also contains LTR-like sequences that are related to exogenous FeLV LTRs. In this study, we evaluated the cellular gene transactivational potential of these endogenous FeLV LTR sequences. Unlike their exogenous FeLV counterparts, neither nearly full-length endogenous FeLV molecular clones (CFE-6 and CFE-16) nor their isolated LTRs were able to activate collagenase IV gene orMCP-1expression in transient transfection assays. We had also demonstrated previously that production of an RNA transcript from exogenous FeLV LTRs correlates with their transactivational activity. In the present study, we demonstrate that the endogenous FeLV LTRs do not generate LTR-specific RNA transcripts in the feline embryo fibroblast cell line AH927. Furthermore, infection of AH927 cells by an exogenous FeLV subgroup A virus did not induce production of such LTR-specific transcripts from the endogenous proviral genomes, although the LTR-specific transcripts from the exogenous virus were readily detected. Finally, LTR-specific transcripts were not generated in BALB/3T3 cells transiently transfected with isolated CFE-6 LTR, in contrast to transfections with LTRs from exogenous viruses. Our data thus suggest that the inability of endogenous FeLV LTRs in gene transactivation is not due to cell line specificity or presence of any upstream inhibitorycis-acting element. Endogenous, nonleukemogenic FeLV LTRs, therefore, do not transactivate cellular gene expression, and this property appears to be specific to exogenous, leukemogenic FeLVs.
Human Immunodeficiency Virus Env-Independent Infection of Human CD4
−
Cells
Authors: Pang Shen , Yu Duan , An Dong Sung , Baldwin Gayle C. , Xie Yiming , Poon Betty , Chow Yen-Hung , Park No-Hee , Chen Irvin S. Y.
Published: July 2002

CD4
−
epithelial cells covering mucosal surfaces serve as the primary barrier to prevent human immunodeficiency virus type 1 (HIV-1) infection. We used HIV-1 vectors carrying the enhanced green fluorescent protein gene as a reporter gene to demonstrate that HIV-1 can infect some CD4
−
human epithelial cell lines with low but significant efficiencies. Importantly, HIV-1 infection of these cell lines is independent of HIV-1 envelope proteins. The Env-independent infection of CD4
−
cells by HIV-1 suggests an alternative pathway for HIV-1 transmission. Even on virions bearing Env, a neutralizing antibody directed against gp120 is incapable of neutralizing the infection of these cells, thus raising potential implications for HIV-1 vaccine development.
Normal T-Cell Turnover in Sooty Mangabeys Harboring Active Simian Immunodeficiency Virus Infection
Authors: Chakrabarti Lisa A. , Lewin Sharon R. , Zhang Linqi , Gettie Agegnehu , Luckay Amara , Martin Louis N. , Skulsky Eva , Ho David D. , Cheng-Mayer Cecilia , Marx Preston A.
Published: July 2002

Sooty mangabeys naturally infected with simian immunodeficiency virus (SIV) remain healthy though they harbor viral loads comparable to those in rhesus macaques that progress to AIDS. To assess the immunologic basis of disease resistance in mangabeys, we compared the effect of SIV infection on T-cell regeneration in both monkey species. Measurement of the proliferation marker Ki-67 by flow cytometry showed that mangabeys harbored proliferating T cells at a level of 3 to 4% in peripheral blood irrespective of their infection status. In contrast, rhesus macaques demonstrated a naturally high fraction of proliferating T cells (7%) that increased two- to threefold following SIV infection. Ki-67+T cells were predominantly CD45RA−, indicating increased proliferation of memory cells in macaques. Quantitation of an episomal DNA product of T-cell receptor α rearrangement (termed α1 circle) showed that the concentration of recent thymic emigrants in blood decreased with age over a 2-log unit range in both monkey species, consistent with age-related thymic involution. SIV infection caused a limited decrease of α1 circle numbers in mangabeys as well as in macaques. Dilution of α1 circles by T-cell proliferation likely contributed to this decrease, since α1 circle numbers and Ki-67+fractions correlated negatively. These findings are compatible with immune exhaustion mediated by abnormal T-cell proliferation, rather than with early thymic failure, in SIV-infected macaques. Normal T-cell turnover in SIV-infected mangabeys provides an explanation for the long-term maintenance of a functional immune system in these hosts.
Readministration of Adenovirus Vector in Nonhuman Primate Lungs by Blockade of CD40-CD40 Ligand Interactions
Authors: Chirmule Narendra , Raper Steven E. , Burkly Linda , Thomas David , Tazelaar John , Hughes Joseph V. , Wilson James M.
Published: July 2002

The interaction between CD40 on B cells and CD40 ligand (CD40L) on activated T cells is important for B-cell differentiation in T-cell-dependent humoral responses. We have extended our previous murine studies of CD40-CD40L in adenoviral vector-mediated immune responses to rhesus monkeys. Primary immune responses to adenoviral vectors and the ability to readminister vector were studied in rhesus monkeys in the presence or absence of a transient treatment with a humanized anti-CD40 ligand antibody (hu5C8). Adult animals were treated with hu5C8 at the time vector was instilled into the lung. Immunological analyses demonstrated suppression of adenovirus-induced lymphoproliferation and cytokine responses (interleukin-2 [IL-2], gamma interferon, IL-4, and IL-10) in hu5C8-treated animals. Animals treated with hu5C8 secreted adenovirus-specific immunoglobulin M (IgM) levels comparable to control animals, but did not secrete IgA or develop neutralizing antibodies; consequently, the animals could be readministered with adenovirus vector expressing alkaline phosphatase. A second study was designed to examine the long-term effects on immune functions of a short course of hu5C8. Acute hu5C8 treatment resulted in significant and prolonged inhibition of the adenovirus-specific humoral response well beyond the time hu5C8 effects were no longer significant. These studies demonstrate the potential of hu5C8 as an immunomodulatory regimen to enable administration of adenoviral vectors, and they advocate testing this model in humans.
Chimeric Matrix Proteins Encoded by Defective Proviruses with Large Internal Deletions in Human T-Cell Leukemia Virus Type 1-Infected Humans
Authors: Morozov Vladimir A. , Lagaye Sylvie , Taylor Graham P. , Matutes Estella , Weiss Robin A.
Published: July 2002

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia/lymphoma (ATLL), HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and other diseases. The mechanisms of virus pathogenesis are still obscure. The occurrence of defective proviruses in HTLV-1-infected cell lines and the peripheral blood mononuclear cells (PBMC) of infected individuals is a frequent feature of virus infection. We detected defective proviruses with large internal deletions in PBMC from ATLL and HAM/TSP patients and in asymptomatic HTLV-1 carriers. Seventeen PCR-amplified defective proviruses were sequenced, and three types of deletions were found. Besides truncated MA and the 5′ end of the genome, truncated CA, truncated SU, and more frequently truncated TM linked to the pX region were detected. Reverse transcription-PCR analysis of PBMC from ATLL patients and asymptomatic carriers also revealed RNA transcripts with large internal deletions. Analysis of two RT-PCR cDNA clones confirmed a Gag-TM-pX structure of the transcripts. Most defective proviruses contained numerous internal stop codons, but some were capable of coding for the truncated MA linked to a variable out-of-frame peptide. Cloned defective proviruses with long open reading frames were subjected to in vitro transcription-translation followed by radioimmunoprecipitation, which showed expression of chimeric proteins between 8 and 12 kDa. Possible roles of defective proviruses and chimeric proteins are discussed, although there is no firm association with pathogenesis.
Deletion of the Cytoplasmic Tail of the Fusion Protein of the Paramyxovirus Simian Virus 5 Affects Fusion Pore Enlargement
Authors: Dutch Rebecca Ellis , Lamb Robert A.
Published: July 2002

The fusion (F) protein of the paramxyovirus simian parainfluenza virus 5 (SV5) promotes virus-cell and cell-cell membrane fusion. Previous work had indicated that removal of the SV5 F protein cytoplasmic tail (F Tail− or FΔ19) caused a block in fusion promotion at the hemifusion stage. Further examination has shown that although the F Tail− mutant is severely debilitated in promotion of fusion as measured by using two reporter gene assays and is debilitated in the formation of syncytia relative to the wild-type F protein, the F Tail− mutant is capable of promoting the transfer of small aqueous dyes. These data indicate that F Tail− is fully capable of promoting formation of small fusion pores. However, enlargement of fusion pores is debilitated, suggesting that either the cytoplasmic tail of the F protein plays a direct role in pore expansion or that it interacts with other components which control pore growth.
Common Antiviral Cytotoxic T-Lymphocyte Epitope for Diverse Arenaviruses
Authors: Oldstone Michael B. A. , Lewicki Hanna , Homann Dirk , Nguyen Christophe , Julien Sylvianne , Gairin Jean Edouard
Published: July 2002

Members of the
Arenaviridae
family have been isolated from mammalian hosts in disparate geographic locations, leading to their grouping as Old World types (i.e., lymphocytic choriomeningitis virus [LCMV], Lassa fever virus [LFV], Mopeia virus, and Mobala virus) and New World types (i.e., Junin, Machupo, Tacaribe, and Sabia viruses) (C. J. Peters, M. J. Buchmeier, P. E. Rollin, and T. G. Ksiazek, p. 1521–1551,
in
B. N. Fields, D. M. Knipe, and P. M. Howley [ed.], Fields virology, 3rd ed., 1996; P. J. Southern, p. 1505–1519,
in
B. N. Fields, D. M. Knipe, and P. M. Howley [ed.], Fields virology, 3rd ed., 1996). Several types in both groups—LFV, Junin, Machupo, and Sabia viruses—cause severe and often lethal human diseases. By sequence comparison, we noted that eight Old World and New World arenaviruses share several amino acids with the nucleoprotein (NP) that consists of amino acids (aa) 118 to 126 (NP 118–126) (RPQASGVYM) of LCMV that comprise the immunodominant cytotoxic T-lymphocyte (CTL) epitope for
H-2

d

mice (32). This L
d
-restricted epitope constituted >97% of the total bulk CTLs produced in the specific antiviral or clonal responses of
H-2

d

BALB mice. NP 118–126 of the Old World arenaviruses LFV, Mopeia virus, and LCMV and the New World arenavirus Sabia virus bound at high affinity to L
d
. The primary
H-2

d

CTL anti-LCMV response as well as that of a CTL clone responsive to LCMV NP 118–126 recognized target cells coated with NP 118–126 peptides derived from LCMV, LFV, and Mopeia virus but not Sabia virus, indicating that a common functional NP epitope exists among Old World arenaviruses. Use of site-specific amino acid exchanges in the NP CTL epitope among these arenaviruses identified amino acids involved in major histocompatibility complex binding and CTL recognition.
The Ribosome Binding Site of Hepatitis C Virus mRNA
Authors: Lytle J. Robin , Wu Lily , Robertson Hugh D.
Published: July 2002

Hepatitis C virus (HCV) infects an estimated 170 million people worldwide, the majority of whom develop a chronic infection which can lead to severe liver disease, and for which no generally effective treatment yet exists. A promising target for treatment is the internal ribosome entry site (IRES) of HCV, a highly conserved domain within a highly variable RNA. Never before have the ribosome binding sites of any IRES domains, cellular or viral, been directly characterized. Here, we reveal that the HCV IRES sequences most closely associated with 80S ribosomes during protein synthesis initiation are a series of discontinuous domains together comprising by far the largest ribosome binding site yet discovered.
Temporal Regulation of Herpes Simplex Virus Type 2 VP22 Expression and Phosphorylation
Authors: Geiss Brian J. , Tavis John E. , Metzger Lisa M. , Leib David A. , Morrison Lynda A.
Published: July 2002

The VP22 protein of herpes simplex virus type 2 (HSV-2) is a major component of the virion tegument. Previous work with HSV-1 indicated that VP22 is phosphorylated during infection, and phosphorylation may play a role in modulating VP22 localization in infected cells. It is not clear, however, when phosphorylation occurs in infected cells or how it is regulated. Less is known about the synthesis and phosphorylation of HSV-2 VP22. To study the complete biosynthetic history of HSV-2 VP22, we generated a monoclonal antibody to the carboxy terminus of VP22. Using immunoprecipitation and Western blot analyses, we show that HSV-2 VP22 can be found in three distinct isoforms in infected cells, two of which are phosphorylated. Like HSV-1 VP22, HSV-2 VP22 is synthesized ca. 4 h after infection, and the isoform later incorporated into virions is hypophosphorylated. In addition, we demonstrate for the first time (i) that newly synthesized VP22 is phosphorylated rapidly after synthesis, (ii) that this phosphorylation occurs in a virus-dependent manner, (iii) that the HSV-2 kinase UL13 is capable of inducing phosphorylation of VP22 in the absence of other viral proteins, (iv) that phosphorylated VP22 is very stable in infected cells, (v) that phosphorylated isoforms of VP22 are gradually dephosphorylated late in infection to produce the virion tegument form, and (vi) that this dephosphorylation occurs independently of viral DNA replication or virion assembly. These results indicate that HSV-2 VP22 is a stable protein that undergoes highly regulated, virus-dependent phosphorylation events in infected cells.
Protection against Tetanus by Needle-Free Inoculation of Adenovirus-Vectored Nasal and Epicutaneous Vaccines
Authors: Shi Zhongkai , Zeng Mingtao , Yang Guang , Siegel Felix , Cain Laura J. , van Kampen Kent R. , Elmets Craig A. , Tang De-Chu C.
Published: July 2002

The effectiveness of vaccination programs would be enhanced greatly through the availability of vaccines that can be administered simply and, preferably, painlessly without the need for timed booster injections. Tetanus is a prime example of a disease that is readily preventable by vaccination but remains a major threat to public health due to the problems associated with administration of the present vaccine. Here we show that a protective immune response against live
Clostridium tetani
infection in mice can be elicited by an adenovirus vector encoding the tetanus toxin C fragment when administered as a nasal or epicutaneous vaccine. The results suggest that these vaccination modalities would be effective needle-free alternatives. This is the first demonstration that absorption of a small number of vectored vaccines into the skin following topical application of a patch can provide protection against live bacteria in a disease setting.
Human Immunodeficiency Virus Type 1 Nef Selectively Associates with a Catalytically Active Subpopulation of p21-Activated Kinase 2 (PAK2) Independently of PAK2 Binding to Nck or β-PIX
Authors: Renkema G. Herma , Manninen Aki , Saksela Kalle
Published: July 2002

We have recently identified the Nef-associated serine-threonine kinase (NAK) as the p21-activated kinase 2 (PAK2). Here we have taken advantage of the possibility to manipulate the functional properties of NAK by transfecting PAK2 cDNA or its mutant derivatives in order to further characterize the Nef-NAK complex. To exclude the possibility that some Nef variants might interact with PAK1 instead of PAK2, we also examined the identity of NAK complexed with divergent human immunodeficiency virus type 1 HIV-1 Nef proteins. All tested Nef proteins, including SF2, NL4-3, BH10, and HAN-2, associated with PAK2 but not with PAK1. By exchanging different regions between these two PAK proteins, the selective ability of PAK2 to associate with Nef could be mapped to the carboxy-terminal part of its regulatory domain. Binding of PAK2 with the adapter protein Nck or β-PIX was found to be dispensable for the assembly of the Nef-PAK2 complex, whereas an intact Cdc42-Rac1 interactive binding motif was required. Most importantly, we found that NAK represented a distinct subpopulation of the total cellular PAK2 characterized by a high specific kinase activity. Thus, although only a small fraction of cellular PAK2 could be found in complex with Nef, NAK represented a major part of cellular PAK2 activity.
Analysis of p53 Inactivation in a Human T-Cell Leukemia Virus Type 1 Tax Transgenic Mouse Model
Authors: Portis Toni , Grossman William J. , Harding John C. , Hess Jay L. , Ratner Lee
Published: July 2002

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia/lymphoma (ATLL). The HTLV-1 Tax protein has been strongly linked to oncogenesis and is considered to be the transforming protein of this virus. A Tax transgenic mouse model was utilized to study the contribution of p53 inactivation to Tax-mediated tumorigenesis. These mice develop primary, peripheral tumors consisting of large granular lymphocytic (LGL) cells, which also infiltrate the lymph nodes, bone marrow, spleen, liver, and lungs. Primary Tax-induced tumors and tumor-derived cell lines exhibited functional inactivation of the p53 apoptotic pathway; such tumors and tumor cell lines were resistant to an apoptosis-inducing stimulus. In contrast,
p53
mutations in tumors were found to be associated with secondary organ infiltration. Three of four identified mutations inhibited transactivation and apoptosis induction activities in vitro. Furthermore, experiments which involved mating Tax transgenic mice with p53-deficient mice demonstrated minimal acceleration in initial tumor formation, but significantly accelerated disease progression and death in mice heterozygous for
p53
. These studies suggest that functional inactivation of p53 by HTLV-1 Tax, whether by mutation or another mechanism, is not critical for initial tumor formation, but contributes to late-stage tumor progression.
Exchange of the Basic Domain of Human Immunodeficiency Virus Type 1 Rev for a Polyarginine Stretch Expands the RNA Binding Specificity, and a Minimal Arginine Cluster Is Required for Optimal RRE RNA Binding Affinity, Nuclear Accumulation, and
trans
-Activation
Authors: Nam Yong-Suk , Petrovic Ana , Jeong Kyu-Shik , Venkatesan Sundararajan
Published: July 2002

The Rev regulatory protein of human immunodeficiency virus (HIV) facilitates the nuclear export of unspliced and partially spliced HIV RNAs. Using a Rev:MS2 phage coat protein fusion that could be targeted to bind and activate the Rev-responsive element (RRE) RNA or heterologous MS2 phage operator RNA, we analyzed the role(s) of the arginine-rich RNA binding domain in RNA binding and transactivation. The arginine-rich domain could be functionally replaced by a stretch of nine arginines. However, polyarginine substitutions expanded the RNA binding specificity of the resultant mutant Rev protein. Polyarginine insertions in place of residues 24 to 60 that excised the RNA binding and oligomerization domains of Rev preserved the activation for MS2 RNA, but not for the RRE. A nine-arginine insertion outside of the natural context of the Rev nuclear localization signal domain was incompatible with activation of either RNA target. Insertions of fewer than eight arginines impaired RRE activation. Interrupted lysine clusters and disruption of the arginine stretch with lysine or neutral residues resulted in a similar phenotype. Some of these mutants with a null phenotype for RRE activated the heterologous MS2 RNA target. Under steady-state conditions, mutants that preserved the Rev response for RRE RNA localized to the nuclei; those with poor or no Rev response accumulated mostly in the cytoplasm. Many of the cytoplasmically resident derivatives became nuclear when leptomycin B (LMB) treatment inhibited nuclear export of nuclear export signal-containing proteins. Mutants that had a null activation potential for either RNA target were particularly resistant to LMB treatment. Abbreviated nuclear residence times and differences in RRE binding affinity may have compromised their activation potential for RRE. High-affinity binding to MS2 RNA through the intact coat protein was sufficient to overcome the short nuclear residence times and to facilitate MS2 activation by some derivatives.
AIDS Vaccination Studies Using an Ex Vivo Feline Immunodeficiency Virus Model: Reevaluation of Neutralizing Antibody Levels Elicited by a Protective and a Nonprotective Vaccine after Removal of Antisubstrate Cell Antibodies
Authors: Giannecchini Simone , Del Mauro Daniela , Matteucci Donatella , Bendinelli Mauro
Published: July 2002

In the feline immunodeficiency virus system, immunization with a fixed-infected-cell vaccine conferred protection against virulent homologous challenge but the immune effectors involved remained elusive. In particular, few or no neutralizing antibodies were detected in sera from vaccinated cats. Here we show that, when preadsorbed with selected feline cells, the same sera revealed clearly evident virus-neutralizing activity. Because high titers of neutralizing antibody in cell-adsorbed sera from 23 cats immunized with fixed-infected-cell or whole-inactivated-virus vaccines correlated with protection, it is likely that they were more important for protection than formerly realized. In vitro, the fixed-cell vaccine efficiently removed neutralizing antibody from immune sera while the whole-inactivated-virus vaccine was much less effective.
Long-Term Circulation of Vaccine-Derived Poliovirus That Causes Paralytic Disease
Authors: Cherkasova Elena A. , Korotkova Ekaterina A. , Yakovenko Maria L. , Ivanova Olga E. , Eremeeva Tatyana P. , Chumakov Konstantin M. , Agol Vadim I.
Published: July 2002

Successful implementation of the global poliomyelitis eradication program raises the problem of vaccination against poliomyelitis in the posteradication era. One of the options under consideration envisions completely stopping worldwide the use of the Sabin vaccine. This strategy is based on the assumption that the natural circulation of attenuated strains and their derivatives is strictly limited. Here, we report the characterization of a highly evolved derivative of the Sabin vaccine strain isolated in a case of paralytic poliomyelitis from a 7-month-old immunocompetent baby in an apparently adequately immunized population. Analysis of the genome of this isolate showed that it is a double (type 1-type 2-type 1) vaccine-derived recombinant. The number of mutations accumulated in both the type 1-derived and type 2-derived portions of the recombinant genome suggests that both had diverged from their vaccine predecessors ∼2 years before the onset of the illness. This fact, along with other recent observations, points to the possibility of long-term circulation of Sabin vaccine strain derivatives associated with an increase in their neurovirulence. Comparison of genomic sequences of this and other evolved vaccine-derived isolates reveals some general features of natural poliovirus evolution. They include a very high preponderance and nonrandom distribution of synonymous substitutions, conservation of secondary structures of important
cis
-acting elements of the genome, and an apparently adaptive character of most of the amino acid mutations, with only a few of them occurring in the antigenic determinants. Another interesting feature is a frequent occurrence of tripartite intertypic recombinants with either type 1 or type 3 homotypic genomic ends.
Organic Reactivity Control by Means of Neighboring Groups and Organometallics. A Personal Account
Authors: Kočovský Pavel
Published: July 2002

This review summarizes the main topics of our research and covers the period of the last 15 years. The prime interest is focused on various ways of controlling the regio- and stereoselectivity of selected organic reactions, in particular electrophilic additions, cleavage of cyclopropane rings, and allylic substitutions by means of neighboring groups and/or transition and non-transition metals. In the first part, the factors governing the course of electrophilic additions are assessed, culminating in the formulation of selection rules for the reactivity of cyclohexene systems, and in a concise synthesis of the natural cardioactive drug, strophanthidin. These studies also contribute to a better understanding of the mechanisms of electrophilic additions. The second part describes recent developments in the stereo- and regiocontrolled cleavage of cyclopropane rings by non-transition metals (Tl and Hg), and the reactivity and transmetalation (with Pd) of the primary products. This methodology has resulted in novel routes to unique polycyclic structures, and will have synthetic applications in the near future. Evidence for the stereospecific "corner" cleavage of the cyclopropane ring has been provided for the first time for Tl and later for Hg. The third part deals with transition metal-catalyzed allylic substitution. Evidence for a new "syn" mechanism for the formation of the intermediate (π-allyl)palladium complex has been provided, which runs counter to the generally accepted "anti" mechanism. A novel method for a Pd-catalyzed allylic oxidation has been developed and employed in the synthesis of natural sesquiterpenes. The increasing importance of transition and non-transition metals for synthetic organic chemistry is demonstrated by their unique reactivity in a number of the papers included in this review.
Synthesis of Oligo(ethylene glycol) Derivatives
Authors: Hosgören Halil , Colak Nureddin , Öztürkmen Nermin
Published: July 2002

Oligo(ethylene glycol) derivatives were obtained by the reaction of oligo(ethylene glycol) chlorohydrins with epichlorohydrin using H2SO4 catalyst and subsequent treatment with NaOH in ether solution. The structure of the obtained products was characterized by 13C NMR and IR spectra.
The Synthesis and Some Properties of N,N'-Bis(3-alkoxyphenyl)ureas
Authors: Gregáň Fridrich , Kettmann Viktor , Csöllei Jozef , Novomeský Pavol
Published: July 2002

We prepared N,N'-bis(3-alkoxyphenyl)ureas IIa - IIh by adding of water to 3-alkoxyphenylisocyanates I in the presence of sodium hydroxide according to the literature.
Synthesis of New 2-Substituted 4-(2-Furyl)-4H-pyrans
Authors: Ilavský Dušan , Bruncko Milan , Marchalín Štefan , Zalibera Lubomír
Published: July 2002

Substituted 2-amino-3-cyano-4H-pyrans represent important precursors in syntheses of condensed heterocycles containing a 4H-pyran nucleus. In the context of studies of reactivity of 2-amino-5-acetyl-4-(5-X-2-furyl)-3-cyano-6-methyl-4H-pyrans we were interested in the possibility of preparing 2-aryl(heteroaryl)methyleneamino-4H-pyrans III.
Carbonylative Reduction of Nitrophenols to Aminophenols
Authors: Macho Vendelín , Kučera Milan , Králik Milan
Published: July 2002

Two- or three-component catalysts composed of (i) sulfur or sulfur compound (H2S, CS2, COS, Na2S), (ii) basic additive (triethylamine, CH3ONa, Na2S), and usually (iii) vanadium(V) compounds (e.g. NH4VO3) were found to catalyze efficiently the reaction of CO + H2O with isomeric nitrophenols to give the corresponding aminophenols. The reaction proceeds smoothly at 398 and 483 K and initial pressure of 7 MPa, and its rate increases from 2- to 4-nitrophenol. The selectivity to aminophenols exceeding 96 per cent was obtained at the water to nitrophenol molar ratio higher than 5. The solvents such as methanol and dioxane ensured better contact of the reactants, which was necessary for achievement of such a high selectivity. The effectiveness of the sulfur components (based on the S content) is expressed by the following sequence: S : CS2 : Na2S : H2 S : COS = 1 : 1.2 : 2.5 : 10 : 11. The reaction takes place also under the reduced CO pressure to 0.1 - 0.35 MPa. Formation of side products and mechanism of the reaction are discussed.
Off-Line SFE of Bis(2-ethylhexyl) Phthalate from PVC Materials
Authors: Ventura Karel , Kovář Richard , Mikešová Milena , Karásek Pavel , Vejrosta Jiří
Published: July 2002

Off-line supercritical fluid extraction with pure CO2 was used to isolate and quantify bis(2-ethylhexyl) phthalate (DIOP) from PVC samples. Two modes of analyte trapping, onto an inner solid surface of a fused silica capillary (i.d. 0.5 mm) and direct bubbling through a bulk liquid (2-propanol), have been compared. Collected samples were analyzed by high performance liquid chromatography with UV detection. Quantitative recovery of over 95% was obtained in spite of limited operating parameters of home-made SFE device (maximum of pressure 30 MPa and of temperature 70 °C) for samples containing 10 or less wt.% of DIOP.
Dynamics of Electrodiffusion Friction Probes. II. Shape-Dependent Impedance
Authors: Wein Ondřej , Sobolík Václav , Tihon Jaroslav
Published: July 2002

The existing linear (impedance) theory of frequency response is corrected and generalized for electrodiffusion friction probes of any shape. Special attention is paid to the dynamic calibration of real electrodiffusion probes with uncertain geometry by the potentiostatic (voltage-step) transient method.
Comparison of the Brillouin-Wigner Coupled Cluster Theory with the Standard Coupled Cluster Theory. Cancellation of Disconnected Terms in the Brillouin-Wigner Coupled Cluster Theory
Authors: Mášik Jozef , Hubač Ivan
Published: July 2002

In our article (Hubac I., Neogrady P.: Phys. Rev. A 50, 4558 (1994)) we have developed size-extensive Brillouin-Wigner coupled cluster (BWCC) theory. We have shown that the BWCC theory is fully equivalent to the standard coupled cluster theory. However, the BWCC theory does not obey the so-called linked cluster theorem. In this article we discuss the cancellation of disconnected terms in the BWCC theory.
A Facile Synthesis of Carboxamides by Dehydration Condensation Between Free Carboxylic Acids and Amines Using O,O'-Di(2-pyridyl) Thiocarbonate as a Coupling Reagent
Authors: Shiina Isamu , Saitoh Katsuyuki , Nakano Masakazu , Suenaga Yoshihito , Mukaiyama Teruaki
Published: July 2002

Carboxamides are prepared in high yields by dehydration condensation between nearly equimolar amounts of free carboxylic acids and amines both of which involve secondary or tertiary alkyl substituted ones with O,O'-di(2-pyridyl) thiocarbonate, a coupling reagent, in the presence of a catalytic amount of 4-(dimethylamino)pyridine.
Solid-Phase Traceless Synthesis of Selected Nitrogen-Containing Heterocyclic Compounds. The Encore Technique for Directed Sorting of Modular Solid Support
Authors: Krchňák Viktor , Smith Jennifer , Vágner Josef
Published: July 2002

The acid lability of electron-rich N-benzylanilines has been exploited in a linker for the traceless solid-phase synthesis of benzimidazoles, 2-aminobenzimidazoles, quinoxalinones and tetrahydroquinoxalines. The target compound precursors were assembled on a solid-phase support derivatized with either a benzylamine or a benzhydrylamine linker. Exposure to an acidic reagent caused cleavage of the C(benzyl)-N(aniline) bond, releasing the product with only a hydrogen atom on the descending nitrogen. The Encore technique for directed sorting on SynPhase Lanterns has been developed and applied to combinatorial synthesis of generic drug discovery libraries.
Exact moment of a gastrocnemius muscle strain captured on video
Authors: Orchard J W , Alcott E , James T , Farhart P , Portus M , Waugh S R
Published: July 2002

A left gastrocnemius strain was sustained by an elite cricket batsman while he was taking off to run. The exact moment of injury, captured by a camera in the middle stump, appears to correspond to the sudden appearance of a deficit in the gastrocnemius muscle, seen through the player's trousers. The strain occurred when the entire body weight was on the left foot with the centre of mass well in front of the leg. The injury probably occurred close to the time when the gastrocnemius complex was moving from an eccentric to an isometric phase.
Positive reform of tuna farm diving in South Australia in response to government intervention
Authors: Whyte P , Doolette D J , Gorman D F , Craig D S
Published: July 2002

OBJECTIVES

Much of the tuna harvested in South Australia since 1990 has involved “farming” techniques requiring the use of divers. From 1993 to 1995, 17 divers from this industry were treated for decompression illness (DCI). In response, the State Government introduced corrective strategies. A decrease in the number of divers presenting for treatment was subsequently recorded. Consequently, the hypothesis was tested that the government intervention resulted in a decrease in the incidence of DCI in the industry and an improved clinical outcome of divers with DCI.

METHODS

The incidence of treated DCI in tuna farm divers was estimated from the number of divers with DCI treated and the number of dives undertaken extrapolated from a survey of the industry in 1997–8. General health was measured in the tuna farm diving population by a valid and reliable self assessment questionnaire. The outcome of the divers treated for DCI was analysed with a modified clinical severity scoring system.

RESULTS

The apparent incidence of treated DCI has decreased in tuna farm divers since the government intervention. The evidence supports a truly decreased incidence rather than underreporting. The general health of the tuna farm divers was skewed towards the asymptomatic end of the range, although health scores indicative of DCI were reported after 1.7% of the dives that did not result in recognised DCI. The clinical outcome of the divers treated since the intervention has improved, possibly because of earlier recognition of the disease and hence less time spent diving while having DCI.

CONCLUSIONS

The government intervention in the tuna industry in South Australia has resulted in a reduced incidence of DCI in the industry.
Pitfalls in the interpretation of common biochemical tests
Authors: Ayling Ruth M
Published: July 2002

This review considers some of the more common problems in the interpretation of the results of biochemical tests and, where possible, highlights ways in which errors can be identified or avoided.
Are adolescents attempting to buy cigarettes on the internet?
Authors: Unger Jennifer B , Rohrbach Louise Ann , Ribisl Kurt M
Published: July 2002

OBJECTIVE

To assess the prevalence of, and demographic and smoking behaviour correlates of, attempting to purchase cigarettes via the internet among adolescent current smokers.

METHODS

A representative sample of 17 181 10th and 12th grade California students completed a written questionnaire on tobacco related attitudes and behaviour during the 1999-2000 school year. Logistic regression analyses were used to determine the variables associated with attempting to purchase cigarettes on the internet.

RESULTS

Among youth under 18 years of age who were current smokers (n = 1689), 2.2% (95% confidence interval 1.5% to 2.9%) reported attempting to purchase cigarettes on the internet. Attempted internet purchases were more likely among younger respondents, males, frequent smokers, and respondents reporting lower perceived availability of tobacco products from retail and social sources.

CONCLUSIONS

Few adolescent smokers in California surveyed during the 1999-2000 school year had attempted to obtain cigarettes from the internet. As internet access increases, future studies should examine whether internet cigarette vendors sell to minors and whether adolescents are purchasing cigarettes on the internet.
Mongoven, Biscoe & Duchin: destroying tobacco control activism from the inside
Authors: Carter Stacy M
Published: July 2002

Mongoven, Biscoe & Duchin, a specialist firm based in Washington DC, has honed a niche as expert intelligence gatherers, helping tobacco companies such as Philip Morris and RJ Reynolds to damage tobacco control efforts, including the WHO's Framework Convention on Tobacco Control.