bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genetic Analysis of the
Sinorhizobium meliloti
BacA Protein: Differential Effects of Mutations on Phenotypes
Authors: LeVier Kristin , Walker Graham C. 
Published: July 2002

ABSTRACT

Sinorhizobium meliloti
strains lacking BacA function are impaired in symbiosis with alfalfa host plants and display altered sensitivities to a number of compounds relative to wild-type strains. With the goal of finding clues to the currently unknown biological function(s) of BacA, we carried out a genetic analysis to determine which amino acids are critical for protein function and to attempt to ascertain whether the multiple phenotypes that result from a
bacA-
null allele were the result of a common cause or whether BacA has multiple functions. We have created a set of 20 site-directed mutants in which selected individual amino acids in
bacA
were replaced with glycine residues. The resulting mutants were characterized to determine how the various amino acid changes affected a number of phenotypes associated with loss of BacA function. Mutants H165G, W182G, D198G, and R284G had null phenotypes for all functions assayed, while mutants W57G, S83G, S231G, and K350G were indistinguishable from wild-type strains. The remaining 12 site-directed mutants demonstrate mixed phenotypic characteristics and fall into a number of distinctly different groups. These observations may be consistent with a role for BacA in multiple, nonoverlapping functions.
H2O2-Forming NADH Oxidase with Diaphorase (Cytochrome) Activity fromArchaeoglobus fulgidus
Authors: Reed David W. , Millstein Jack , Hartzell Patricia L. 
Published: July 2002

ABSTRACTAn enzyme exhibiting NADH oxidase (diaphorase) activity was isolated from the hyperthermophilic sulfate-reducing anaerobeArchaeoglobus fulgidus. N-terminal sequence of the protein indicates that it is coded for by open reading frame AF0395 in theA. fulgidusgenome. The gene AF0395 was cloned and its product was purified fromEscherichia coli. Like the native NADH oxidase (NoxA2), the recombinant NoxA2 (rNoxA2) has an apparent molecular mass of 47 kDa, requires flavin adenine dinucleotide for activity, has NADH-specific activity, and is thermostable. Hydrogen peroxide is the product of bivalent oxygen reduction by rNoxA2 with NADH. The rNoxA2 is an oxidase with diaphorase activity in the presence of electron acceptors such as tetrazolium and cytochromec. During purification NoxA2 remains associated with the enzyme responsible ford-lactate oxidation, thed-lactate dehydrogenase (Dld), and the genes encoding NoxA2 and Dld are in the same transcription unit. Together these results suggest that NADH oxidase may be involved in electron transfer reactions resulting in sulfate respiration.
Characterization of Flagellum Gene Families of Methanogenic Archaea and Localization of Novel Flagellum Accessory Proteins
Authors: Thomas Nikhil A. , Jarrell Ken F. 
Published: July 2002

ABSTRACTArchaeal flagella are unique motility structures, and the absence of bacterial structural motility genes in the complete genome sequences of flagellated archaeal species suggests that archaeal flagellar biogenesis is likely mediated by novel components. In this study, a conserved flagellar gene family from each ofMethanococcus voltae,Methanococcus maripaludis,Methanococcus thermolithotrophicus, andMethanococcus jannaschiihas been characterized. These species possess multiple flagellin genes followed immediately by eight known and supposed flagellar accessory genes,flaCDEFGHIJ. Sequence analyses identified a conserved Walker box A motif in the putative nucleotide binding proteins FlaH and FlaI that may be involved in energy production for flagellin secretion or assembly. Northern blotting studies demonstrated that all the species have abundant polycistronic mRNAs corresponding to some of the structural flagellin genes, and in some cases several flagellar accessory genes were shown to be cotranscribed with the flagellin genes. Cloned flagellar accessory genes ofM. voltaewere successfully overexpressed as His-tagged proteins inEscherichia coli.These recombinant flagellar accessory proteins were affinity purified and used as antigens to raise polyclonal antibodies for localization studies. Immunoblotting of fractionatedM. voltaecells demonstrated that FlaC, FlaD, FlaE, FlaH, and FlaI are all present in the cell as membrane-associated proteins but are not major components of isolated flagellar filaments. Interestingly,flaDwas found to encode two proteins, each translated from a separate ribosome binding site. These protein expression data indicate for the first time that the putative flagellar accessory genes ofM. voltae, and likely those of other archaeal species, do encode proteins that can be detected in the cell.
Role for Vitamin B
12
in Light Induction of Gene Expression in the Bacterium
Myxococcus xanthus
Authors: Cervantes María , Murillo Francisco J. 
Published: July 2002

ABSTRACT

A light-inducible promoter (P
B
) drives the
carB
operon (carotenoid genes) of the bacterium
Myxococcus xanthus
. A gene encoding a regulator of carotenoid biosynthesis was identified by studying mutant strains carrying a transcriptional fusion to P
B
and deletions in three candidate genes. Our results prove that the identified gene, named
carA
, codes for a repressor of the P
B
promoter in the dark. They also show that the
carA
gene product does not participate in the light activation of two other promoters connected with carotenoid synthesis or its regulation in
M. xanthus
. CarA is a novel protein consisting of a DNA-binding domain of the family of MerR helix-turn-helix transcriptional regulators, directly joined to a cobalamin-binding domain. In support of this, we report here that the presence of vitamin B
12
or some other cobalamin derivatives is absolutely required for activation of the P
B
promoter by light.
Overexpression of the
Escherichia coli sugE
Gene Confers Resistance to a Narrow Range of Quaternary Ammonium Compounds
Authors: Chung Yong Joon , Saier Milton H. 
Published: July 2002

ABSTRACT

SugE of
Escherichia coli
, first identified as a suppressor of
groEL
mutations but a member of the small multidrug resistance family, has not previously been shown to confer a drug resistance phenotype. We show that high-level expression of
sugE
leads to resistance to a subset of toxic quaternary ammonium compounds.
Phylogenetic Analysis of
Lacazia loboi
Places This Previously Uncharacterized Pathogen within the Dimorphic Onygenales
Authors: Herr Roger A. , Tarcha Eric J. , Taborda Paulo R. , Taylor John W. , Ajello Libero , Mendoza Leonel 
Published: July 2002

ABSTRACT

Lacazia loboi
is the last of the classical fungal pathogens to remain a taxonomic enigma, primarily because it has resisted cultivation and only causes cutaneous and subcutaneous infections in humans and dolphins in the New World tropics. To place it in the evolutionary tree of life, as has been done for the other enigmatic human pathogens
Pneumocystis carinii
and
Rhinosporidium seeberi
, we amplified its 18S small-subunit ribosomal DNA (SSU rDNA) and 600 bp of its chitin synthase-2 gene. Our phylogenetic analysis indicated that
L. loboi
is the sister taxon of the human dimorphic fungal pathogen
Paracoccidioides brasiliensis
and that both species belong with the other dimorphic fungal pathogens in the order Onygenales. The low nucleotide variation among three
P. brasiliensis
18S SSU rDNA sequences contrasts with the surprising amount of nucleotide differences between the two sequences of
L. loboi
used in this study, suggesting that the nucleic acid epidemiology of this hydrophilic pathogen will be rewarding.
Molecular Characterization of
Cryptosporidium
Isolates Obtained from Humans in France
Authors: Guyot K. , Follet-Dumoulin A. , Lelièvre E. , Sarfati C. , Rabodonirina M. , Nevez G. , Cailliez J. C. , Camus D. , Dei-Cas E. 
Published: July 2002

ABSTRACT

Cryptosporidium parvum
is usually considered the agent of human cryptosporidiosis. However, only in the last few years, molecular biology-based methods have allowed the identification of
Cryptosporidium
species and genotypes, and only a few data are available from France. In the present work, we collected samples of whole feces from 57 patients from France (11 immunocompetent patients, 35 human immunodeficiency virus [HIV]-infected patients, 11 immunocompromised but non-HIV-infected patients) in whom
Cryptosporidium
oocysts were recognized by clinical laboratories. A fragment of the
Cryptosporidium
18S rRNA gene encompassing the hypervariable region was amplified by PCR and sequenced. The results revealed that the majority of the patients were infected with cattle (29 of 57) or human (18 of 57) genotypes of
Cryptosporidium parvum
. However, a number of immunocompromised patients were infected with
C. meleagridis
(3 of 57),
C. felis
(6 of 57), or a new genotype of
C. muris
(1 of 57). This is the first report of the last three species of
Cryptosporidium
in humans in France. These results indicate that immunocompromised individuals are susceptible to a wide range of
Cryptosporidium
species and genotypes.
Rapid Detection of Herpes Simplex Virus DNA in Genital Ulcers by Real-Time PCR Using SYBR Green I Dye as the Detection Signal
Authors: Aldea Carmen , Alvarez Carmen P. , Folgueira Lola , Delgado Rafael , Otero Joaquín R. 
Published: July 2002

ABSTRACT
We have evaluated a real-time PCR procedure based on the LightCycler technology for rapid detection of herpes simplex virus (HSV) in genital lesions. Two sets of primers, corresponding to the thymidine kinase and DNA polymerase regions, were used for the amplification reactions in separate capillaries containing the SYBR Green I dye as detection signal. In 28 of 118 samples (24%), HSV was isolated by conventional cell culture. All cell culture-positive samples were also positive by real-time PCR. Six additional cell culture-negative samples were positive by PCR with both sets of primers. Total processing time was less than 3 h. Real-time PCR using SYBR Green I as detection signal is a sensitive procedure for the rapid diagnosis of HSV in genital lesions.
Household Transmission of
Streptococcus pneumoniae
among Siblings with Acute Otitis Media
Authors: Shimada Jun , Yamanaka Noboru , Hotomi Muneki , Suzumoto Masaki , Sakai Akihiro , Ubukata Kimiko , Mitsuda Toshihiro , Yokota Shumpei , Faden Howard 
Published: July 2002

ABSTRACT

Nasopharyngeal transmission of
Streptococcus pneumoniae
was evaluated among 23 siblings with acute otitis media (AOM). Restriction fragment length polymorphism revealed that the nasopharyngeal strains were identical between siblings in 12 of 13 clusters of AOM experienced in 11 families. This study demonstrated person-to-person transmission of
S. pneumoniae
, especially drug-resistant strains, among siblings with AOM.
Effects of Deletion or Stringent Repression of the H3L Envelope Gene on Vaccinia Virus Replication
Authors: da Fonseca Flávio G. , Wolffe Elizabeth J. , Weisberg Andrea , Moss Bernard 
Published: July 2002

ABSTRACT
The C-terminal membrane anchor protein encoded by the H3L open reading frame of vaccinia virus is located on the surfaces of intracellular mature virions. To investigate the role of the H3L protein, we constructed deletion (vH3Δ) and inducible (vH3i) null mutants. The H3L protein was not detected in lysates of cells infected with vH3Δ or vH3i in the absence of inducer. Under these conditions, plaques were small and round instead of large and comet shaped, indicative of decreased virus replication or cell-to-cell spread. The mutant phenotype was correlated with reduced yields of infectious intra- and extracellular virus in one-step growth experiments. The defect in vH3i replication could not be attributed to a role of the H3L protein in virus binding, internalization, or any event prior to late gene expression. Electron microscopic examination of cells infected with vH3Δ or vH3i in the absence of inducer revealed that virion assembly was impaired, resulting in a high ratio of immature to mature virus forms with an accumulation of crescent membranes adjacent to granular material and DNA crystalloids. The absence of the H3L protein did not impair the membrane localization of virion surface proteins encoded by the A27L, D8L, and L1R genes. The wrapping of virions and actin tail formation were not specifically blocked, but there was an apparent defect in low-pH-mediated syncytium formation that could be attributed to decreased virus particle production. The phenotypes of the H3L deletion and repression mutants were identical to each other but differed from those produced by null mutations of genes encoding other vaccinia virus membrane components.
Alphaherpesvirus Proteins Related to Herpes Simplex Virus Type 1 ICP0 Affect Cellular Structures and Proteins
Authors: Parkinson Jane , Everett Roger D. 
Published: July 2002

ABSTRACT
The herpes simplex virus type 1 (HSV-1) immediate-early protein ICP0 interacts with several cellular proteins and induces the proteasome-dependent degradation of others during infection. In this study we show that ICP0 is required for the proteasome-dependent degradation of the ND10 protein Sp100 and, as with the other target proteins, the ICP0 RING finger domain is essential. Further, comparison of the kinetics and ICP0 domain requirements for the degradation of PMI and Sp100 suggests that a common mechanism is involved. Homologues of ICP0 are encoded by other members of the alphaherpesvirus family. These proteins show strong sequence homology to ICP0 within the RING finger domain but limited similarity elsewhere. Using transfection assays, we have shown that all the ICP0 homologues that we tested have significant effects on the immunofluorescence staining character of at least one of the proteins destabilized by ICP0, and by using a recombinant virus, we found that the equine herpesvirus ICP0 homologue induced the proteasome-dependent degradation of endogenous CENP-C and modified forms of PML and Sp100. However, in contrast to ICP0, the homologue proteins had no effect on the distribution of the ubiquitin-specific protease USP7 within the cell, consistent with their lack of a USP7 binding domain. We also found that ICP0 by itself could induce the abrogation of SUMO-1 conjugation and then the proteasome-dependent degradation of unmodified exogenous PML in transfected cells, thus demonstrating that other HSV-1 proteins are not required. Surprisingly, the ICP0 homologues were unable to cause these effects. Overall, these data suggest that the members of the ICP0 family of proteins may act via a similar mechanism or pathway involving their RING finger domain but that their intrinsic activities and effects on endogenous and exogenous proteins differ in detail.
Lymphatic Dissemination and Comparative Pathology of Recombinant Measles Viruses in Genetically Modified Mice
Authors: Mrkic Branka , Odermatt Bernhard , Klein Michael A. , Billeter Martin A. , Pavlovic Jovan , Cattaneo Roberto 
Published: July 2002

ABSTRACT
The dissemination of the Edmonston measles virus (Ed-MV) vaccine strain was studied with genetically modified mice defective for the alpha/beta interferon receptor and expressing human CD46 with human-like tissue specificity and efficiency. A few days after intranasal infection, macrophages expressing Ed-MV RNA were detected in the lungs, in draining lymph nodes, and in the thymus. In lymph nodes, large syncytia which stained positive for viral RNA and for macrophage surface marker proteins were found and apoptotic cell death was monitored. In the thymus, smaller syncytia which stained positive for macrophage and dendritic cell markers were detected. Thus, macrophages appear to be the main vectors for dissemination of MV infection in these mice; human macrophages may have a similar function in the natural host. We then compared the pathogenicities of two recombinant viruses lacking the C or V nonstructural proteins to that of the parental strain, Ed-MV. These viruses were less effective in spreading through the lymphatic system and, unlike Ed-MV, were not detected in the liver. After intracerebral inoculation the recombinant viruses caused lethal disease less often than Ed-MV and induced distinctive patterns of gliosis and inflammation. Ed-MV was reisolated from brain tissue, but its derivatives were not. C- and V-defective viruses should be considered as more-attenuated MV vaccine candidates.
Replication of Lengthened Moloney Murine Leukemia Virus Genomes Is Impaired at Multiple Stages
Authors: Shin Nam-Hee , Hartigan-O'Connor Dennis , Pfeiffer Julie K. , Telesnitsky Alice 
Published: July 2002

ABSTRACT
It has been assumed that RNA packaging constraints limit the size of retroviral genomes. This notion of a retroviral “headful” was tested by examining the ability of Moloney murine leukemia virus genomes lengthened by 4, 8, or 11 kb to participate in a single replication cycle. Overall, replication of these lengthened genomes was 5- to 10-fold less efficient than that of native-length genomes. When RNA expression and virion formation, RNA packaging, and early stages of replication were compared, long genomes were found to complete each step less efficiently than did normal-length genomes. To test whether short RNAs might facilitate the packaging of lengthy RNAs by heterodimerization, some experiments involved coexpression of a short packageable RNA. However, enhancement of neither long vector RNA packaging nor long vector DNA synthesis was observed in the presence of the short RNA. Most of the proviruses templated by 12 and 16 kb vectors appeared to be full length. Most products of a 19.2-kb vector contained deletions, but some integrated proviruses were around twice the native genome length. These results demonstrate that lengthy retroviral genomes can be packaged and that genome length is not strictly limited at any individual replication step. These observations also suggest that the lengthy read-through RNAs postulated to be intermediates in retroviral transduction can be packaged directly without further processing.
Basic Residues in Human Immunodeficiency Virus Type 1 Nucleocapsid Promote Virion Assembly via Interaction with RNA
Authors: Cimarelli Andrea , Sandin Sara , Höglund Stefan , Luban Jeremy 
Published: July 2002

ABSTRACTRetroviral Gag polyproteins drive virion assembly by polymerizing to form a spherical shell that lines the inner membrane of nascent virions. Deletion of the nucleocapsid (NC) domain of the Gag polyprotein disrupts assembly, presumably because NC is required for polymerization. Human immunodeficiency virus type 1 NC possesses two zinc finger motifs that are required for specific recognition and packaging of viral genomic RNA. Though essential, zinc fingers and genomic RNA are not required for virion assembly. NC promiscuously associates with cellular RNAs, many of which are incorporated into virions. It has been hypothesized that Gag polymerization and virion assembly are promoted by nonspecific interaction of NC with RNA. Consistent with this model, we found an inverse relationship between the number of NC basic residues replaced with alanine and NC's nonspecific RNA-binding activity, Gag's ability to polymerize in vitro and in vivo, and Gag's capacity to assemble virions. In contrast, mutation of NC's zinc fingers had only minor effects on these properties.
Biology of E1-Deleted Adenovirus Vectors in Nonhuman Primate Muscle
Authors: Zoltick Philip W. , Chirmule Narendra , Schnell Michael A. , Gao Guang-ping , Hughes Joseph V. , Wilson James M. 
Published: July 2002

ABSTRACTAdenovirus vectors have been studied as vehicles for gene transfer to skeletal muscle, an attractive target for gene therapies for inherited and acquired diseases. In this setting, immune responses to viral proteins and/or transgene products cause inflammation and lead to loss of transgene expression. A few studies in murine models have suggested that the destructive cell-mediated immune response to virally encoded proteins of E1-deleted adenovirus may not contribute to the elimination of transgene-expressing cells. However, the impact of immune responses following intramuscular administration of adenovirus vectors on transgene stability has not been elucidated in larger animal models such as nonhuman primates. Here we demonstrate that intramuscular administration of E1-deleted adenovirus vector expressing rhesus monkey erythropoietin or growth hormone to rhesus monkeys results in generation of a Th1-dependent cytotoxic T-cell response to adenovirus proteins. Transgene expression dropped significantly over time but was still detectable in some animals after 6 months. Systemic levels of adenovirus-specific neutralizing antibodies were generated, which blocked vector readministration. These studies indicate that the cellular and humoral immune response generated to adenovirus proteins, in the context of transgenes encoding self-proteins, hinders long-term transgene expression and readministration with first-generation vectors.
Spindle Cell Conversion by Kaposi's Sarcoma-Associated Herpesvirus: Formation of Colonies and Plaques with Mixed Lytic and Latent Gene Expression in Infected Primary Dermal Microvascular Endothelial Cell Cultures
Authors: Ciufo Dolores M. , Cannon Jennifer S. , Poole Lynn J. , Wu Frederick Y. , Murray Paul , Ambinder Richard F. , Hayward Gary S. 
Published: July 2002

ABSTRACT

Angiogenic Kaposi's sarcoma (KS) skin lesions found in both AIDS and non-AIDS patients are universally associated with infection by the presumed causative agent, known as KS-associated herpesvirus (KSHV) or human herpesvirus 8. KSHV genomes expressing latent state virus-encoded mRNAs and the LANA1 (latent nuclear antigen 1) protein are consistently present in spindle-like tumor cells that are thought to be of endothelial cell origin. Although the KSHV lytic cycle can be induced in rare latently infected primary effusion lymphoma (PEL) cell lines, the ability to transmit or assay infectious KSHV has so far eluded investigators. Here, we demonstrate that infection with supernatant virions derived from three different tetradecanoyl phorbol acetate-induced PEL cell lines can induce cultured primary human dermal microvascular endothelial cells (DMVEC) to form colonies of proliferating latently infected spindle-shaped cells, all of which express the KSHV-encoded LANA1 protein. Although their initial infectivity varied widely (JSC1 > > BC3 > BCP1), virions from all three cell lines produced distinctive spindle cell colonies and plaques without affecting the contact-inhibited cobblestone-like phenotype of adjacent uninfected DMVEC. Each infected culture could also be expanded into a completely spindloid persistently infected culture displaying aggregated swirls of spindle cells resembling those in KS lesions. Formation of new colonies and plaques was inhibited in the presence of phosphonoacetic acid or gangciclovir, but these antiherpesvirus agents had little effect on the propagation of already latently infected spindloid cultures. In persistently infected secondary cultures, patches of up to 10% of the spindloid cells constitutively expressed several early viral lytic cycle proteins, and 1 to 2% of the cells also formed typical herpesvirus DNA replication compartments, displayed cytopathic rounding effects, and expressed late viral antigens. We conclude that
de novo
KSHV infection induces a spindle cell conversion phenotype in primary DMVEC cultures that is directly associated with latent state expression of the LANA1 protein. However, these cultures also spontaneously reactivate to produce an unusual combination of both latent and productive but slow lytic cycle infection. The formation of spindle cell colonies and plaques in DMVEC cultures provides for the first time a quantitative assay for directly measuring the infectivity of KSHV virion preparations.