bluuebunny/crossref_metadata_2025_split huggingface dataset:
Bacillus subtilis
SMC Is Required for Proper Arrangement of the Chromosome and for Efficient Segregation of Replication Termini but Not for Bipolar Movement of Newly Duplicated Origin Regions
Authors: Graumann Peter L. 
Published: July 2002

ABSTRACT

SMC protein is required for chromosome condensation and for the faithful segregation of daughter chromosomes in
Bacillus subtilis
. The visualization of specific sites on the chromosome showed that newly duplicated origin regions in growing cells of an
smc
mutant were able to segregate from each other but that the location of origin regions was frequently aberrant. In contrast, the segregation of replication termini was impaired in
smc
mutant cells. This analysis was extended to germinating spores of an
smc
mutant. The results showed that during germination, newly duplicated origins, but not termini, were able to separate from each other in the absence of SMC. Also, DAPI (4′,6′-diamidino-2-phenylindole) staining revealed that chromosomes in germinating spores were able to undergo partial or complete replication but that the daughter chromosomes were blocked at a late stage in the segregation process. These findings were confirmed by time-lapse microscopy, which showed that after duplication in growing cells the origin regions underwent rapid movement toward opposite poles of the cell in the absence of SMC. This indicates that SMC is not a required component of the mitotic motor that initially drives origins apart after their duplication. It is also concluded that SMC is needed to maintain the proper layout of the chromosome in the cell and that it functions in the cell cycle after origin separation but prior to complete segregation or replication of daughter chromosomes. It is proposed here that chromosome segregation takes place in at least two steps: an SMC-independent step in which origins move apart and a subsequent SMC-dependent step in which newly duplicated chromosomes condense and are thereby drawn apart.
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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACTThe 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

ABSTRACT
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

ABSTRACTWe 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

ABSTRACT

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

ABSTRACTSooty 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

ABSTRACTThe 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

ABSTRACT
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

ABSTRACTThe 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.