bluuebunny/crossref_metadata_2025_split huggingface dataset:
Organization and Expression of the Polynucleotide Phosphorylase Gene (
pnp
) of
Streptomyces
: Processing of
pnp
Transcripts in
Streptomyces antibioticus
Authors: Bralley Patricia , Jones George H. 
Published: May 2004

ABSTRACT

We have examined the expression of
pnp
encoding the 3′-5′-exoribonuclease, polynucleotide phosphorylase, in
Streptomyces antibioticus
. We show that the
rpsO
-
pnp
operon is transcribed from at least two promoters, the first producing a readthrough transcript that includes both
pnp
and the gene for ribosomal protein S15 (
rpsO
) and a second, P
pnp
, located in the
rpsO-pnp
intergenic region. Unlike the situation in
Escherichia coli
, where observation of the readthrough transcript requires mutants lacking RNase III, we detect readthrough transcripts in wild-type
S. antibioticus
mycelia. The P
pnp
transcriptional start point was mapped by primer extension and confirmed by RNA ligase-mediated reverse transcription-PCR, a technique which discriminates between 5′ ends created by transcription initiation and those produced by posttranscriptional processing. Promoter probe analysis demonstrated the presence of a functional promoter in the intergenic region. The P
pnp
sequence is similar to a group of promoters recognized by the extracytoplasmic function sigma factors, sigma-R and sigma-E. We note a number of other differences in
rspO-pnp
structure and function between
S. antibioticus
and
E. coli
. In
E. coli
,
pnp
autoregulation and cold shock adaptation are dependent upon RNase III cleavage of an
rpsO-pnp
intergenic hairpin. Computer modeling of the secondary structure of the
S. antibioticus
readthrough transcript predicts a stem-loop structure analogous to that in
E. coli
. However, our analysis suggests that while the readthrough transcript observed in
S. antibioticus
may be processed by an RNase III-like activity, transcripts originating from P
pnp
are not. Furthermore, the
S. antibioticus rpsO-pnp
intergenic region contains two open reading frames. The larger of these,
orfA
, may be a pseudogene. The smaller open reading frame,
orfX
, also observed in
Streptomyces coelicolor
and
Streptomyces avermitilis
, may be translationally coupled to
pnp
and the gene downstream from
pnp
, a putative protease.
Rusty, Jammed, and Well-Oiled Hinges: Mutations Affecting the Interdomain Region of FliG, a Rotor Element of the
Escherichia coli
Flagellar Motor
Authors: Van Way Susan M. , Millas Stephanos G. , Lee Aaron H. , Manson Michael D. 
Published: May 2004

ABSTRACT

The FliG protein is a central component of the bacterial flagellar motor. It is one of the first proteins added during assembly of the flagellar basal body, and there are 26 copies per motor. FliG interacts directly with the Mot protein complex of the stator to generate torque, and it is a crucial player in switching the direction of flagellar rotation from clockwise (CW) to counterclockwise and vice versa. A primarily helical linker joins the N-terminal assembly domain of FliG, which is firmly attached to the FliF protein of the MS ring of the basal body, to the motility domain that interacts with MotA/MotB. We report here the results of a mutagenic analysis focused on what has been called the hinge region of the linker. Residue substitutions in this region generate a diversity of phenotypes, including motors that are strongly CW biased, infrequent switchers, rapid switchers, and transiently or permanently paused. Isolation of these mutants was facilitated by a “sensitizing” mutation (E232G) outside of the hinge region that was accidentally introduced during cloning of the chromosomal
fliG
gene into our vector plasmid. This mutation partially interferes with flagellar assembly and accentuates the defects associated with mutations that by themselves have little phenotypic consequence. The effects of these mutations are analyzed in the context of a conformational-coupling model for motor switching and with respect to the structure of the C-terminal 70% of FliG from
Thermotoga maritima
.
Complexity of Gas Vesicle Biogenesis inHalobacteriumsp. Strain NRC-1: Identification of Five New Proteins
Authors: Shukla Hem Dutt , DasSarma Shiladitya 
Published: May 2004

ABSTRACTThe genome ofHalobacteriumsp. strain NRC-1 contains a large gene cluster,gvpMLKJIHGFEDACNO, that is both necessary and sufficient for the production of buoyant gas-filled vesicles. Due to the resistance of gas vesicles to solubilization, only the major gas vesicle protein GvpA and a single minor protein, GvpC, were previously detected. Here, we used immunoblotting analysis to probe for the presence of gas vesicle proteins corresponding to five additionalgvpgene products. Polyclonal antisera were raised in rabbits against LacZ-GvpF, -GvpJ, and -GvpM fusion proteins and against synthetic 15-amino-acid peptides from GvpG and -L. Immunoblotting analysis was performed on cell lysates of wild-typeHalobacteriumsp. strain NRC-1, gas vesicle-deficient mutants, and purified gas vesicles, after purification of LacZ fusion antibodies on protein A and β-galactosidase affinity columns. Our results show the presence of five new gas vesicle proteins (GvpF, GvpG, GvpJ, GvpL, and GvpM), bringing the total number of proteins identified in the organelles to seven. Two of the new gas vesicle proteins are similar to GvpA (GvpJ and GvpM), and two proteins contain predicted coiled-coil domains (GvpF and GvpL). GvpL exhibited a multiplet ladder on sodium dodecyl sulfate-polyacrylamide gels indicative of oligomerization and self-assembly. We discuss the possible functions of the newly discovered gas vesicle proteins in biogenesis of these unique prokaryotic flotation organelles.
Arsenic Resistance inHalobacteriumsp. Strain NRC-1 Examined by Using an Improved Gene Knockout System
Authors: Wang Gejiao , Kennedy Sean P. , Fasiludeen Sabeena , Rensing Christopher , DasSarma Shiladitya 
Published: May 2004

ABSTRACTThe genome sequence ofHalobacteriumsp. strain NRC-1 encodes genes homologous to those responsible for conferring resistance to arsenic. These genes occur on both the large extrachromosomal replicon pNRC100 (arsADRCandarsR2M) and on the chromosome (arsB). We studied the role of thesearsgenes in arsenic resistance genetically by construction of gene knockouts. Deletion of thearsADRCgene cluster in aHalobacteriumNRC-1 Δura3 strain resulted in increased sensitivity to arsenite and antimonite but not arsenate. In contrast, knockout of the chromosomalarsBgene did not show significantly increased sensitivity to arsenite or arsenate. We also found that knockout of thearsMgene produced sensitivity to arsenite, suggesting a second novel mechanism of arsenic resistance involving a putative arsenite(III)-methyltransferase. These results indicate thatHalobacteriumsp. strain NRC-1 contains an arsenite and antimonite extrusion system with significant differences from bacterial counterparts. Deletion analysis was facilitated by an improved method for gene knockouts/replacements inHalobacteriumthat relies on both selection and counterselection ofura3using a uracil dropout medium and 5-fluoroorotic acid. The arsenite and antimonite resistance elements were shown to be regulated, with resistance to arsenic in the wild type inducible by exposure to a sublethal concentration of the metal. Northern hybridization and reverse transcription-PCR analyses showed thatarsA,arsD,arsR,arsM,arsC, andarsB, but notarsR2, are inducible by arsenite and antimonite. We discuss novel aspects of arsenic resistance in this halophilic archaeon and technical improvements in our capability for gene knockouts in the genome.
Evaluation of the Kinetic Properties of the Sporulation Protein SpoIIE of
Bacillus subtilis
by Inclusion in a Model Membrane
Authors: Searls Tim , Chen Xingyong , Allen Stephanie , Yudkin Michael D. 
Published: May 2004

ABSTRACT

Starvation induces
Bacillus subtilis
to initiate a developmental process (sporulation) that includes asymmetric cell division to form the prespore and the mother cell. The integral membrane protein SpoIIE is essential for the prespore-specific activation of the transcription factor σ
F
, and it also has a morphogenic activity required for asymmetric division. An increase in the local concentration of SpoIIE at the polar septum of
B. subtilis
precedes dephosphorylation of the anti-anti-sigma factor SpoIIAA in the prespore. After closure and invagination of the asymmetric septum, phosphatase activity of SpoIIE increases severalfold, but the reason for this dramatic change in activity has not been determined. The central domain of SpoIIE has been seen to self-associate (I. Lucet et al., EMBO J. 19:1467-1475, 2000), suggesting that activation of the C-terminal PP2C-like phosphatase domain might be due to conformational changes brought about by the increased local concentration of SpoIIE in the sporulating septum. Here we report the inclusion of purified SpoIIE protein into a model membrane as a method for studying the effect of local concentration in a lipid bilayer on activity. In vitro assays indicate that the membrane-bound enzyme maintains dephosphorylation rates similar to the highly active micellar state at all molar ratios of protein to lipid. Atomic force microscopy images indicate that increased local concentration does not lead to self-association.
Salmonella enterica
Serovar Typhi Strains from Which SPI7, a 134-Kilobase Island with Genes for Vi Exopolysaccharide and Other Functions, Has Been Deleted
Authors: Nair Satheesh , Alokam Suneetha , Kothapalli Sushma , Porwollik Steffan , Proctor Emily , Choy Carmen , McClelland Michael , Liu Shu-Lin , Sanderson Kenneth E. 
Published: May 2004

ABSTRACT

Salmonella enterica
serovar Typhi has a 134-kb island of DNA identified as salmonella pathogenicity island 7 (SPI7), inserted between
pheU
and ′
pheU
(truncated), two genes for tRNA
Phe
. SPI7 has genes for Vi exopolysaccharide, for type IVB pili, for putative conjugal transfer, and for sopE bacteriophage. Pulsed-field gel electrophoresis following digestion with the endonuclease I-CeuI, using DNA from a set of 120 wild-type strains of serovar Typhi assembled from several sources, identified eight strains in which the I-CeuI G fragment, which contains SPI7, had a large deletion. In addition, agglutination tests with Vi antiserum and phage typing with Vi phages show that all eight strains are Vi negative. We therefore tested these strains for deletion of SPI7 by multiplex PCR, by microarray analysis, and by sequencing of PCR amplicons. Data show that seven of the eight strains are precise deletions of SPI7: a primer pair flanking SPI7 results in a PCR amplicon containing a single
pheU
gene; microarrays show that all SPI7 genes are deleted. Two of the strains produce amplicons which have A derived from
pheU
at bp 27, while five have C derived from ′
pheU
at this position; thus, the position of the crossover which results in the deletion can be inferred. The deletion in the eighth strain, TYT1669, removes 175 kb with junction points in genes STY4465 and STY4664; the left junction of SPI7 and adjacent genes, as well as part of SPI7 including the
viaB
operon for Vi exopolysaccharide, was removed, while the right junction of SPI7 was retained. We propose that these deletions occurred during storage following isolation.
Isolation and Characterization of a Generalized Transducing Phage for
Pseudomonas aeruginosa
Strains PAO1 and PA14
Authors: Budzik Jonathan M. , Rosche William A. , Rietsch Arne , O'Toole George A. 
Published: May 2004

ABSTRACT

A temperate, type IV pilus-dependent, double-stranded DNA bacteriophage named DMS3 was isolated from a clinical strain of
Pseudomonas aeruginosa
. A clear-plaque variant of this bacteriophage was isolated. DMS3 is capable of mediating generalized transduction within and between
P. aeruginosa
strains PA14 and PAO1, thus providing a useful tool for the genetic analysis of
P. aeruginosa
.
−1 Frameshifting at a CGA AAG Hexanucleotide Site Is Required for Transposition of Insertion Sequence IS
1222
Authors: Mejlhede Nina , Licznar Patricia , Prère Marie-Françoise , Wills Norma M. , Gesteland Raymond F. , Atkins John F. , Fayet Olivier 
Published: May 2004

ABSTRACT

The discovery of programmed −1 frameshifting at the hexanucleotide shift site CGA_AAG, in addition to the classical X_XXY_YYZ heptanucleotide shift sequences, prompted a search for instances among eubacterial insertion sequence elements. IS
1222
has a CGA_AAG shift site. A genetic analysis revealed that frameshifting at this site is required for transposition.
The etiology of severe anemia in a village and a periurban area in Mali
Authors: Dicko Alassane , Klion Amy D. , Théra Mahamadou A. , Sagara Issaka , Yalcouyé Daniel , Niambele Mohamed B. , Sogoba Moussa , Dolo Guimogo , Dao Adama , Diallo Dapa A. , Doumbo Ogobara K. , Miller Louis H. 
Published: May 2004

Abstract
Severe anemia is one of the major complications of malaria in Africa. We studied 2 populations, one in a village and the second in a periurban area in Mali, to understand the preventable factors in the disease. The 2 correlates of disease were parasitemia above 100 000 parasitized red blood cells per microliter (0.1 × 1012/L) and a low baseline hemoglobin level. All cases of moderate to severe anemia occurred in children under 3.2 years of age. Raising the baseline hemoglobin level and lowering peak parasitemia in infants and young children may reduce the incidence of severe anemia resulting from malarial infection. (Blood. 2004;104: 1198-1200)
Tuning the volume of the immune response: strength and persistence of stimulation determine migration and cytokine secretion of dendritic cells
Authors: Luft Thomas , Maraskovsky Eugene , Schnurr Max , Knebel Katja , Kirsch Michael , Görner Martin , Skoda Radek , Ho Anthony D. , Nawroth Peter , Bierhaus Angelika 
Published: May 2004

AbstractMigration to lymph nodes and secretion of cytokines are critical functions of mature dendritic cells (DCs); however, these 2 functions are not necessarily linked. This is the first report showing that quantitative differences in identical signaling pathways determine DC migration and cytokine secretion. Using different polymerized forms of CD40 ligand, we demonstrate that the strength and persistence of CD40 signaling can induce either function. Induction of monocyte-derived DC (MoDC) migration required a weak and transient CD40 signal, whereas strong and persistent CD40 signaling blocked migration and biased toward cytokine secretion. In contrast to MoDCs, CD40 activation of CD1c+ peripheral blood DCs (PBDCs) induced a nonpersistent, intracellular signaling profile resulting in migratory-type DCs unable to secrete interleukin-12p70 (IL-12p70). Extracellular signal-regulated kinase 1/2 (ERK1/2) and p38K activation synergistically mediated cytokine secretion, whereas migration was enhanced by p38K activation but reduced by persistent ERK1/2 activity. This model of signal strength and persistence also applied when stimulating DCs with intact microbes. Thus, a novel concept emerges in which the type of immune response induced by DCs is tuned by the strength and persistence of DC activating signals.
Pleiotropic consequences of Bruton tyrosine kinase deficiency in myeloid lineages lead to poor inflammatory responses
Authors: Mangla Anita , Khare Anupriya , Vineeth Varanasi , Panday Nagesh Narayan , Mukhopadhyay Asok , Ravindran Balachandran , Bal Vineeta , George Anna , Rath Satyajit 
Published: May 2004

AbstractBruton tyrosine kinase (Btk), a non-receptor-associated tyrosine kinase of the Tec family, appears to participate in many myeloid cell functions. We show that macrophages from X-linked immunodeficient (XID) mice lacking functional Btk cannot generate efficient bursts of reactive oxygen intermediates (ROIs). The induction of apoptotic cell death by inflammatory stimuli is also enhanced in XID macrophages. Phagocytosis of bacterial particles is only marginally affected in them. In vivo, XID mice show reduced severity of inflammatory diseases in models of experimental autoimmune encephalomyelitis (EAE), dextran sulfate sodium (DSS)-induced colitis, and carrageenan-induced acute edema. Also, polymorphonuclear neutrophil granulocytes (PMNs) in XID mice show poor ROI and nitric oxide (NO) induction, along with a reduction in PMN recruitment to peritoneal inflammation. XID mice show reduction in PMN numbers in peripheral blood, and their bone marrow shows a reduction in the numbers of both monocytic and granulocytic lineages, extending to the earliest progenitor populations. Thus, Btk is likely to play a significant role at multiple points during the development and functioning of the myeloid lineages, affecting the outcome of many infectious as well as noninfectious inflammatory events in vivo. (Blood. 2004;104:1191-1197)
Risk factors for syngeneic graft-versus-host disease after adult hematopoietic cell transplantation
Authors: Adams Kristina M. , Holmberg Leona A. , Leisenring Wendy , Fefer Alexander , Guthrie Katherine A. , Tylee Tracy S. , McDonald George B. , Bensinger William I. , Nelson J. Lee 
Published: May 2004

Abstract
Syngeneic graft-versus-host disease (sGVHD) has been described after hematopoietic cell transplantation (HCT) but remains poorly defined. We retrospectively reviewed adult syngeneic HCTs at our center (1980-2002) for sGVHD to investigate incidence, morbidity, and risk factors with a primary focus on parity. Among 119 transplantations, there were 21 cases of biopsy-proven sGVHD. The cumulative incidence was 18%, with multiorgan involvement in 6 cases and 1 death. sGVHD was more frequent when the donor was parous (32%) than nulliparous (9%) or male (13%; P = .03) and when the recipient was parous (31%) than nulliparous (7%) or male (13%; P = .02). Other univariable risk factors included older age (P &lt; .01), busulfan/melphalan/thiotepa conditioning (P &lt; .01), interleukin-2 (P = .02), HLA-A26 (P = .03), and more recent transplantation year (P &lt; .01). Overall, risk factors were similar to those described in GVHD. Although an independent effect of parity could not be completely separated from other factors, donor and recipient pregnancy history merits further investigation.
Killing of human immunodeficiency virus-infected primary T-cell blasts by autologous natural killer cells is dependent on the ability of the virus to alter the expression of major histocompatibility complex class I molecules
Authors: Bonaparte Matthew I. , Barker Edward 
Published: May 2004

AbstractIn the current study, we evaluated whether the capacity of HIV to modulate major histocompatibility complex (MHC) class I molecules has an impact on the ability of autologous natural killer (NK) cells to kill the HIV-infected cells. Analysis of HIV-infected T-cell blasts revealed that the decrease in MHC class I molecules on the infected cell surface was selective. HLA-A and -B were decreased on cells infected with HIV strains that could decrease MHC class I molecules, whereas HLA-C and -E remained on the surface. Blocking the interaction between HLA-C and -E and their corresponding inhibitory receptors increased NK cell killing of T-cell blasts infected with HIV strains that reduced MHC class I molecules. Moreover, we demonstrate that NK cells lacking HLA-C and -E inhibitory receptors kill T-cell blasts infected with HIV strains that decrease MHC class I molecules. In contrast, NK cells are incapable of destroying T-cell blasts infected with HIV strains that were unable to reduce MHC class I molecules. These findings suggest that NK cells lacking inhibitory receptors to HLA-C and -E kill HIV-infected CD4+ T cells, and they indicate that the capacity of NK cells to destroy HIV-infected cells depends on the ability of the virus to modulate MHC class I molecules. (Blood. 2004;104:2087-2094)
Continuous in vivo infusion of interferon-gamma (IFN-γ) preferentially reduces myeloid progenitor numbers and enhances engraftment of syngeneic wild-type cells in Fancc-/- mice
Authors: Li Xiaxin , Yang Yanzhu , Yuan Jin , Hong Ping , Freie Brian , Orazi Attilio , Haneline Laura S. , Clapp D. Wade 
Published: May 2004

AbstractFanconi anemia (FA) is characterized by bone marrow (BM) failure and cancer susceptibility. Identification of the cDNAs of many FA complementation types allows the potential of using gene transfer technology to introduce functional cDNAs as transgenes into autologous stem cells and provide a cure for the BM failure in FA patients. Previous studies in FA murine models and in a phase 1 clinical trial suggest that myelopreparation is required for significant engraftment of exogenous, genetically corrected stem cells. Since myeloid progenitors from Fancc-/- mice and human Fanconi anemia group C protein (FANCC) patients have increased apoptosis in response to interferon γ (IFN-γ) in vitro, we hypothesized that IFN-γ may be useful as a nongenotoxic, myelopreparative conditioning agent. To test this hypothesis, IFN-γ was administered as a continuous infusion to Fancc-/- and wild-type (WT) mice for 1 week. Primitive and mature myeloid lineages were preferentially reduced in IFN-γ-treated Fancc-/- mice. Further, IFN-γ conditioning of Fancc-/- recipients was sufficient as a myelopreparative regimen to allow consistent engraftment of isogenic WT repopulating stem cells. Collectively, these data demonstrate that Fancc-/- hematopoietic cell populations have increased hypersensitivity to IFN-γ in vivo and that IFN-γ conditioning may be useful as a nongenotoxic strategy for myelopreparation in this disorder. (Blood. 2004;104:1204-1209)
CD1d-restricted “NKT” cells and myeloid IL-12 production: an immunological crossroads leading to promotion or suppression of effective anti-tumor immune responses?
Authors: Gumperz Jenny E 
Published: May 2004

AbstractCD1d-restricted T cells are remarkable for their unusual ability to respond to self-antigens and to contribute to both immunostimulatory and immunosuppressive responses. Their effects in different cancer models have appeared contradictory; in some cases, they are linked to the generation of effective tumor clearance, and in others, they seem to contribute to suppression of anti-tumor responses. Recent results suggest CD1d-restricted T cells are involved in critical interactions with myeloid dendritic cells (DCs) that can affect the subsequent course of the immune response, and that factors such as the strength of the antigenic signal and the presence or absence of proinflammatory cytokines may determine the outcome of these interactions. In the presence of a strong antigenic signal, CD1d-restricted T cells induced myeloid DCs to secrete interleukin (IL)-12, and these DCs in turn activated naive T cells to secrete Th1 cytokines. When exposed to the weak antigenic stimulus of self-antigens, CD1d-restricted T cells induced DCs to secrete IL-10 but not IL-12, and these DCs failed to stimulate Th1 cytokine production by naive T cells. In contrast, CD1d-restricted T cells that were stimulated by self-antigens in the presence of IL-12 potently secreted interferon-γ (IFN-γ) and were among the first lymphocytes to become activated in vivo. Hence, CD1d-restricted T cells may promote or prevent effective anti-tumor responses that are mediated by other lymphocytic effector cells by influencing IL-12 production by myeloid DCs and by their own production of early IFN-γ in response to IL-12.