bluuebunny/crossref_metadata_2025 huggingface dataset:
Orc6 Involved in DNA Replication, Chromosome Segregation, and Cytokinesis
Authors: Prasanth Supriya G. , Prasanth Kannanganattu V. , Stillman Bruce
Published: October 2002

Origin recognition complex (ORC) proteins serve as a landing pad for the assembly of a multiprotein prereplicative complex, which is required to initiate DNA replication. During mitosis, the smallest subunit of human ORC, Orc6, localizes to kinetochores and to a reticular-like structure around the cell periphery. As chromosomes segregate during anaphase, the reticular structures align along the plane of cell division and some Orc6 localizes to the midbody before cells separate. Silencing of Orc6 expression by small interfering RNA (siRNA) resulted in cells with multipolar spindles, aberrant mitosis, formation of multinucleated cells, and decreased DNA replication. Prolonged periods of Orc6 depletion caused a decrease in cell proliferation and increased cell death. These results implicate Orc6 as an essential gene that coordinates chromosome replication and segregation with cytokinesis.
An Aligned Stream of Low-Metallicity Clusters in the Halo of the Milky Way
Authors: Yoon Suk-Jin , Lee Young-Wook
Published: October 2002

One of the long-standing problems in modern astronomy is the curious division of Galactic globular clusters, the “Oosterhoff dichotomy,” according to the properties of their RR Lyrae stars. Here, we find that most of the lowest metallicity ([Fe/H] < –2.0) clusters, which are essential to an understanding of this phenomenon, display a planar alignment in the outer halo. This alignment, combined with evidence from kinematics and stellar population, indicates a captured origin from a satellite galaxy. We show that, together with the horizontal-branch evolutionary effect, the factor producing the dichotomy could be a small time gap between the cluster-formation epochs in the Milky Way and the satellite. The results oppose the traditional view that the metal-poorest clusters represent the indigenous and oldest population of the Galaxy.
Biallelic Inactivation of
BRCA2
in Fanconi Anemia
Authors: Howlett Niall G. , Taniguchi Toshiyasu , Olson Susan , Cox Barbara , Waisfisz Quinten , de Die-Smulders Christine , Persky Nicole , Grompe Markus , Joenje Hans , Pals Gerard , Ikeda Hideyuki , Fox Edward A. , D'Andrea Alan D.
Published: October 2002

Fanconi anemia (FA) is a rare autosomal recessive cancer susceptibility disorder characterized by cellular hypersensitivity to mitomycin C (MMC). Six FA genes have been cloned, but the gene or genes corresponding to FA subtypes B and D1 remain unidentified. Here we show that cell lines derived from FA-B and FA-D1 patients have biallelic mutations in
BRCA2
and express truncated BRCA2 proteins. Functional complementation of FA-D1 fibroblasts with wild-type
BRCA2
complementary DNA restores MMC resistance. Our results link the six cloned FA genes with
BRCA1
and
BRCA2
in a common pathway. Germ-line mutation of genes in this pathway may result in cancer risks similar to those observed in families with
BRCA1
or
BRCA2
mutations.
An Archean Impact Layer from the Pilbara and Kaapvaal Cratons
Authors: Byerly Gary R. , Lowe Donald R. , Wooden Joseph L. , Xie Xiaogang
Published: October 2002

The Barberton greenstone belt of South Africa and the eastern Pilbara block of Western Australia provide information about Earth's surface environments between 3.2 and 3.5 billion years ago, including evidence for four large bolide impacts that likely created large craters, deformed the target rocks, and altered the environment. We have obtained identical single-zircon uranium-lead ages of 3470 ± 2 million years ago for the oldest impact events from each craton. These deposits represent a single global fallout layer that is associated with sedimentation by an impact-generated tsunami and in Western Australia is represented by a major erosional unconformity.
Polymer Vesicles
Authors: Discher Dennis E. , Eisenberg Adi
Published: October 2002

Vesicles are microscopic sacs that enclose a volume with a molecularly thin membrane. The membranes are generally self-directed assemblies of amphiphilic molecules with a dual hydrophilic-hydrophobic character. Biological amphiphiles form vesicles central to cell function and are principally lipids of molecular weight less than 1 kilodalton. Block copolymers that mimic lipid amphiphilicity can also self-assemble into vesicles in dilute solution, but polymer molecular weights can be orders of magnitude greater than those of lipids. Structural features of vesicles, as well as properties including stability, fluidity, and intermembrane dynamics, are greatly influenced by characteristics of the polymers. Future applications of polymer vesicles will rely on exploiting unique property-performance relations, but results to date already underscore the fact that biologically derived vesicles are but a small subset of what is physically and chemically possible.
Antiviral Activities of MCC-478, a Novel and Specific Inhibitor of Hepatitis B Virus
Authors: Kamiya Naohiro , Kubota Atsushi , Iwase Yumiko , Sekiya Kouichi , Ubasawa Masaru , Yuasa Satoshi
Published: October 2002

MCC-478 is a newly synthesized 2-amino-6-arylthio-9-phosphonomethoxyethylpurine bis(2,2,2-trifluoroethyl) ester derivative. MCC-478 showed a substantially higher (ca. 80-fold) anti-hepatitis B virus (HBV) activity than that of lamivudine, despite no significant anti-human immunodeficiency virus activity. Since the bis(2,2,2-trifluoroethyl) ester group was used to improve the oral bioavailability of the phosphonomethoxyethylpurine derivatives, two monoester derivatives and one phosphonic acid derivative were also evaluated. It was suggested that these hydrolyzed derivatives, which appeared in animals given MCC-478, have enough anti-HBV activity to contribute to efficacy in vivo. Furthermore, no apparent cytotoxic effects or reductions of mitochondrial DNA content by MCC-478 and its derivatives were observed. These results indicated that MCC-478 may be a new promising anti-HBV agent.
High Incidence of Cefoxitin and Clindamycin Resistance among Anaerobes in Taiwan
Authors: Teng Lee-Jene , Hsueh Po-Ren , Tsai Jui-Chang , Liaw Shwu-Jen , Ho Shen-Wu , Luh Kwen-Tay
Published: October 2002

Susceptibilities to 16 antimicrobial agents were determined by measurement of MICs for 344 isolates of anaerobic bacteria recovered from patients with significant infections. Resistance rates varied among antimicrobial agents and the species tested. The β-lactams were more active in gram-positive than in gram-negative anaerobes. Resistance to meropenem was low (<1%). For β-lactam-β-lactamase inhibitors, piperacillin-tazobactam was most active for all species (resistance, <6%). The rates of resistance to cefoxitin (31 to 65%) and clindamycin (50 to 70%) for non-
Bacteroides
fragilis
species of the
B. fragilis
group were higher than those for
B. fragilis
(4% resistant to cefoxitin and 33% resistant to clindamycin). Among members of
B. fragilis
group,
Bacteroides thetaiotaomicron
was the most resistant to clindamycin (70%) and cefoxitin (65%). Rates of susceptibility to imipenem and metronidazole for
B. fragilis
continue to be high compared to those from a previous study 10 years ago. However, resistance to metronidazole was found recently in five strains of
B. fragilis
. We analyzed the genetic relationships among the metronidazole-resistant
B. fragilis
strains by pulsed-field gel electrophoresis. The metronidazole-resistant
B. fragilis
strains showed genotypic heterogeneity, excluding the dissemination of a single clone.
Antibiotic Pharmacodynamics in Surgical Prophylaxis: an Association between Intraoperative Antibiotic Concentrations and Efficacy
Authors: Zelenitsky Sheryl A. , Ariano Robert E. , Harding Godfrey K. M. , Silverman Richard E.
Published: October 2002

The objective of this study was to characterize the relationship between gentamicin concentrations during surgery and the development of wound infection following colorectal operations. Despite decades of research in surgical prophylaxis, the relationship between intraoperative antibiotic concentrations and postoperative infection and the concentrations required for effective prophylaxis have not been established. A pharmacodynamic analysis was conducted using data from a previous prospective, randomized, double-blind clinical study which compared two dosage regimens of gentamicin plus metronidazole for prophylaxis in connection with elective colorectal surgery. Univariate and multivariate analyses of risk factors for postoperative wound infection were conducted, and the relationship between intraoperative gentamicin concentrations and surgical outcome was characterized. The gentamicin concentration at the time of surgical closure was one of the strongest independent risk factors for infection (P= 0.02), along with the presence of diabetes mellitus (P= 0.02), stoma (P= 0.04), and advanced age (P= 0.05). Gentamicin concentrations at closure of less than 0.5 mg/liter were associated with an infection rate of 80% (representing 8 of 10 patients with concentrations below that level) (P= 0.003). Receiver operating characteristic curve analysis identified a critical closure concentration of 1.6 mg/liter for effective surgical prophylaxis (P= 0.002; sensitivity, 70.8%; specificity, 65.9%). This study provides new and important information on antibiotic pharmacodynamics in surgical prophylaxis. It demonstrates the critical effect of the antibiotic concentration at closure on wound infection and suggests a significant association between the concentration and other well-established risk factors, like the timing of preoperative antibiotic administration and surgery duration.
Development of a Yeast Assay for Rapid Screening of Inhibitors of Human-Derived
Pneumocystis carinii
Dihydrofolate Reductase
Authors: Ma Liang , Jia Qiuyao , Kovacs Joseph A.
Published: October 2002

Human-derived
Pneumocystis carinii
dihydrofolate reductase (DHFR) was expressed in a
Saccharomyces cerevisiae
strain whose growth depends on complementation by this enzyme. We utilized a quantitative assay to measure the sensitivity of this yeast strain to DHFR inhibitors. This assay should be useful for identifying new inhibitors of human-derived
P. carinii
DHFR.
Control of Resistant Pink Bollworm (
Pectinophora gossypiella
) by Transgenic Cotton That Produces
Bacillus thuringiensis
Toxin Cry2Ab
Authors: Tabashnik Bruce E. , Dennehy Timothy J. , Sims Maria A. , Larkin Karen , Head Graham P. , Moar William J. , Carrière Yves
Published: October 2002

Crops genetically engineered to produce
Bacillus thuringiensis
toxins for insect control can reduce use of conventional insecticides, but insect resistance could limit the success of this technology. The first generation of transgenic cotton with
B. thuringiensis
produces a single toxin, Cry1Ac, that is highly effective against susceptible larvae of pink bollworm (
Pectinophora gossypiella
), a major cotton pest. To counter potential problems with resistance, second-generation transgenic cotton that produces
B. thuringiensis
toxin Cry2Ab alone or in combination with Cry1Ac has been developed. In greenhouse bioassays, a pink bollworm strain selected in the laboratory for resistance to Cry1Ac survived equally well on transgenic cotton with Cry1Ac and on cotton without Cry1Ac. In contrast, Cry1Ac-resistant pink bollworm had little or no survival on second-generation transgenic cotton with Cry2Ab alone or with Cry1Ac plus Cry2Ab. Artificial diet bioassays showed that resistance to Cry1Ac did not confer strong cross-resistance to Cry2Aa. Strains with >90% larval survival on diet with 10 μg of Cry1Ac per ml showed 0% survival on diet with 3.2 or 10 μg of Cry2Aa per ml. However, the average survival of larvae fed a diet with 1 μg of Cry2Aa per ml was higher for Cry1Ac-resistant strains (2 to 10%) than for susceptible strains (0%). If plants with Cry1Ac plus Cry2Ab are deployed while genes that confer resistance to each of these toxins are rare, and if the inheritance of resistance to both toxins is recessive, the efficacy of transgenic cotton might be greatly extended.
High-Throughput Methods for Culturing Microorganisms in Very-Low-Nutrient Media Yield Diverse New Marine Isolates
Authors: Connon Stephanie A. , Giovannoni Stephen J.
Published: October 2002

Microbial diversity studies based on the cloning and sequencing of DNA from nature support the conclusion that only a fraction of the microbial diversity is currently represented in culture collections. Out of over 40 known prokaryotic phyla, only half have cultured representatives. In an effort to culture the uncultured phylotypes from oligotrophic marine ecosystems, we developed high-throughput culturing procedures that utilize the concept of extinction culturing to isolate cultures in small volumes of low-nutrient media. In these experiments, marine bacteria were isolated and cultivated at in situ substrate concentrations—typically 3 orders of magnitude less than common laboratory media. Microtiter plates and a newly developed procedure for making cell arrays were employed to raise the throughput rate and lower detection sensitivity, permitting cell enumeration from 200-μl aliquots of cultures with densities as low as 10
3
cells/ml. Approximately 2,500 extinction cultures from 11 separate samplings of marine bacterioplankton were screened over the course of 3 years. Up to 14% of the cells collected from coastal seawater were cultured by this method, which was 14- to 1,400-fold higher than the numbers obtained by traditional microbiological culturing techniques. Among the microorganisms cultured were four unique cell lineages that belong to previously uncultured or undescribed marine
Proteobacteria
clades known from environmental gene cloning studies. These cultures are related to the clades SAR11 (α subclass), OM43 (β subclass), SAR92 (γ subclass), and OM60/OM241 (γ subclass). This method proved successful for the cultivation of previously uncultured marine bacterioplankton that have consistently been found in marine clone libraries.
A Single Ala139-to-Glu Substitution in theRenibacterium salmoninarumVirulence-Associated Protein p57 Results in Antigenic Variation and Is Associated with Enhanced p57 Binding to Chinook Salmon Leukocytes
Authors: Wiens Gregory D. , Pascho Ron , Winton James R.
Published: October 2002

The gram-positive bacteriumRenibacterium salmoninarumproduces relatively large amounts of a 57-kDa protein (p57) implicated in the pathogenesis of salmonid bacterial kidney disease. Antigenic variation in p57 was identified by using monoclonal antibody 4C11, which exhibited severely decreased binding toR. salmoninarumstrain 684 p57 and bound robustly to the p57 proteins of seven otherR. salmoninarumstrains. This difference in binding was not due to alterations in p57 synthesis, secretion, or bacterial cell association. The molecular basis of the 4C11 epitope loss was determined by amplifying and sequencing the two identical genes encoding p57,msa1andmsa2. The 5′ and coding sequences of the 684msa1andmsa2genes were identical to those of the ATCC 33209msa1andmsa2genes except for a single C-to-A nucleotide mutation. This mutation was identified in both themsa1andmsa2genes of strain 684 and resulted in an Ala139-to-Glu substitution in the amino-terminal region of p57. We examined whether this mutation in p57 altered salmonid leukocyte and rabbit erythrocyte binding activities.R. salmoninarumstrain 684 extracellular protein exhibited a twofold increase in agglutinating activity for chinook salmon leukocytes and rabbit erythrocytes compared to the activity of the ATCC 33209 extracellular protein. A specific and quantitative p57 binding assay confirmed the increased binding activity of 684 p57. Monoclonal antibody 4C11 blocked the agglutinating activity of the ATCC 33209 extracellular protein but not the agglutinating activity of the 684 extracellular protein. These results indicate that the Ala139-to-Glu substitution altered immune recognition and was associated with enhanced biological activity ofR. salmoninarum684 p57.
Characterization of ISRgn
1
, a Novel Insertion Sequence of the IS
3
Family Isolated from a Bacteriocin-Negative Mutant of
Ruminococcus gnavus
E1
Authors: Gomez Ana , Ladire Monique , Marcille Francoise , Nardi Michelle , Fons Michel
Published: October 2002

ISRgn
1
, an insertion sequence of the IS3 family, has been identified in the genome of a bacteriocin-negative mutant of
Ruminococcus gnavus
E1. The copy number of ISRgn
1
in
R. gnavus
E1, as well as its distribution among phylogenetically E1-related strains, has been determined. Results obtained suggest that ISRgn
1
is not indigenous to the
R. gnavus
phylogenetic group but that it can transpose in this bacterium.
Worldwide Distribution of
Nitrosococcus oceani
, a Marine Ammonia-Oxidizing γ-Proteobacterium, Detected by PCR and Sequencing of 16S rRNA and
amoA
Genes
Authors: Ward Bess B. , O'Mullan Gregory D.
Published: October 2002

Diversity of cultured ammonia-oxidizing bacteria in the γ-subdivision of the
Proteobacteria
was investigated by using strains isolated from various parts of the world ocean. All the strains were very similar to each other on the basis of the sequences of both the 16S rRNA and ammonia monooxygenase genes and could be characterized as a single species. Sequences were also cloned directly from environmental DNA from coastal Pacific and Atlantic sites, and these sequences represented the first
Nitrosococcus oceani
-like sequences obtained directly from the ocean. Most of the environmental sequences clustered tightly with those of the cultivated strains, but some sequences could represent new species of
Nitrosococcus
. These findings imply that organisms similar to the cultivated
N. oceani
strains have a worldwide distribution.
Coadministration of an Interleukin-12 Gene and aTrypanosoma cruziGene Improves Vaccine Efficacy
Authors: Katae Masaharu , Miyahira Yasushi , Takeda Kazuyoshi , Matsuda Hironori , Yagita Hideo , Okumura Ko , Takeuchi Tsutomu , Kamiyama Tsuneo , Ohwada Akihiko , Fukuchi Yoshinosuke , Aoki Takashi
Published: October 2002

We tested the immunogenicity of twoTrypanosoma cruziantigens injected into mice in the form of DNA vaccine. Immunization with DNA encoding dihydroorotate dehydrogenase did not confer protective immunity in all mouse strains tested. Immunization with DNA encoding trans-sialidase surface antigen (TSSA) protected C57BL/6 (H-2b) mice but not BALB/c (H-2d) or C3H/Hej (H-2k) mice against lethalT. cruziinfection. In vivo depletion of CD4+or CD8+T cells abolished the protective immunity elicited by TSSA gene in C57BL/6 mice. Enzyme-linked immunospot assay with splenocytes fromT. cruzi-infected mice or TSSA gene-vaccinated mice identified anH-2Kb-restricted antigenic peptide, ANYNFTLV. The CD8+-T-cell line specific for this peptide could recognizeT. cruzi-infected cells in vitro and could protect naive mice from lethal infection when adoptively transferred. Coadministration of the interleukin-12 (IL-12) gene with the TSSA gene facilitated the induction of ANYNFTLV-specific CD8+T cells and improved the vaccine efficacy against lethalT. cruziinfection. These results reinforced the utility of immunomodulatory adjuvants such as IL-12 gene for eliciting protective immunity against intracellular parasites by DNA vaccination.
Effect of
katG
Mutations on the Virulence of
Mycobacterium tuberculosis
and the Implication for Transmission in Humans
Authors: Pym Alexander S. , Saint-Joanis Brigitte , Cole Stewart T.
Published: October 2002

The usefulness of isoniazid (INH), a key component of short-course chemotherapy of tuberculosis, is threatened by the emergence of drug-resistant strains of
Mycobacterium tuberculosis
with mutations in the
katG
gene. It is shown here that the most commonly occurring KatG mutation, where Ser 315 is replaced by Thr (S315T), is associated with clinically significant levels of INH resistance. In contrast to another resistant isolate, in which Pro replaces Thr 275, the S315T mutant produces active catalase-peroxidase and is virulent in the mouse model of the disease, indicating that a significant loss of bacterial fitness does not result from this frequent mutation. The implications of this finding for the transmission and reactivation of multidrug-resistant strains of
M. tuberculosis
are severe.
Anthrax Edema Toxin Requires Influx of Calcium for Inducing Cyclic AMP Toxicity in Target Cells
Authors: Kumar Praveen , Ahuja Nidhi , Bhatnagar Rakesh
Published: October 2002

The anthrax edema toxin comprises two proteins: protective antigen and edema factor. Anthrax protective antigen binds to the receptors on the surface of target cells and facilitates the entry of edema factor into these target cells. Edema factor (EF) is an adenylate cyclase that catalyzes the synthesis of cyclic AMP (cAMP) in the cytosol of the host cells. In this study, we examined the requirement of extracellular calcium for anthrax edema toxin-induced toxicity in host cells. The cAMP response generated by edema toxin was analyzed in a variety of cells, including CHO, macrophage-like RAW264.7, human neutrophils, and human lymphocytes. Our investigations reveal that after EF reaches the cell cytosol, a rapid influx of calcium is triggered in the host cell that has a pivotal role in determining the cAMP response of the affected cells. Although the cAMP response generated by edema toxin in different cell types varied in intensity and in the time of initiation, the influx of calcium invariably preceded cAMP accumulation. Agents that blocked the uptake of calcium also inhibited edema toxin-induced accumulation of cAMP in the host cells. This is the first report that demonstrates that edema toxin induces accumulation of cAMP in lymphocytes. By accumulating cAMP, a potent inhibitor of immune cell function, edema toxin may actually be poisoning the immune system and thus facilitating the survival of the bacteria in the host.
Mycobacterium lepraeInfection in Monocyte-Derived Dendritic Cells and Its Influence on Antigen-Presenting Function
Authors: Hashimoto Ken , Maeda Yumi , Kimura Hiroaki , Suzuki Koichi , Masuda Akihiro , Matsuoka Masanori , Makino Masahiko
Published: October 2002

Host defense againstMycobacterium lepraeinfection is chiefly mediated by gamma interferon (IFN-γ)-secreting cytotoxic T cells. Since which antigen-presenting cell populations act to stimulate these T cells is not fully understood, we addressed the role of monocyte-derived dendritic cells (DCs). The DCs phagocytosedM. lepraeand expressed bacterially derived antigens (Ags), such as phenolic glycolipid 1 (PGL-1), in the cytoplasm, as well as on the cell surface. The expression of HLA-ABC and -DR Ags on DCs was down-regulated byM. lepraeinfection, and that of CD86 was up-regulated, but not as fully as byMycobacterium bovisBCG infection. Induction of CD83 expression required a large number ofM. lepraecells. When a multiplicity of infection of >40 was used, the DCs induced a significant proliferative and IFN-γ-producing response in autologous T cells. However, these responses were significantly lower than those induced by BCG- orMycobacterium avium-infected DCs. A CD40-mediated signaling inM. leprae-infected DCs up-regulated the expression of HLA Ags, CD86, and CD83 but did not enhance T-cell-stimulating ability. Therefore,M. leprae-infected DCs are less efficient at inducing T-cell responses. However, when the surface PGL-1 onM. leprae-infected DCs was masked by a monoclonal antibody, the DCs induced enhanced responses in both CD4+- and CD8+-T-cell subsets.M. lepraeis a unique pathogen which remains resistant to DC-mediated T-cell immunity, at least in the early stages of infection.
Simultaneous Expression of Different Variant-Specific Surface Proteins in Single
Giardia lamblia
Trophozoites during Encystation
Authors: Carranza Pedro G. , Feltes Gisela , Ropolo Andrea , Quintana Silvia M. C. , Touz María C. , Luján Hugo D.
Published: October 2002

A novel variant-specific surface protein (VSP) from
Giardia
was identified using the monoclonal antibody 9B10, raised against purified cyst walls. VSP9B10B is preferentially induced during encystation and expressed simultaneously with other VSPs on the surface of encysting trophozoites. These results support the hypothesis that encystation and antigenic variation are processes that are mechanistically related.
Cellular Immunity, but Not Gamma Interferon, Is Essential for Resolution ofBabesia microtiInfection in BALB/c Mice
Authors: Clawson Michael L. , Paciorkowski Natalia , Rajan T. V. , La Vake Carson , Pope Conny , La Vake Morgan , Wikel Stephen K. , Krause Peter J. , Radolf Justin D.
Published: October 2002

A new strain ofBabesia microti(KR-1) was isolated from a Connecticut resident with babesiosis by hamster inoculation and adapted to C3H/HeJ and BALB/c mice. To examine the relative importance of humoral and cellular immunity for the control ofB. microtiinfection, we compared the course of disease in wild-type BALB/c mice with that in BALB/c SCID mice, JHD-null (B-cell-deficient) mice, and T-cell receptor αβ (TCRβ−/−) or gamma interferon (IFN-γ) (IFN-γ−/−) knockout mice following inoculation with the KR-1-strain. SCID mice and TCRαβ knockouts sustained a severe but nonlethal parasitemia averaging 35 to 45% infected erythrocytes. IFN-γ-deficient mice developed a less severe parasitemia but were able to clear the infection. In contrast, in six of eight JHD-null mice, the levels of parasitemia were indistinguishable from those in the wild-type animals. These data indicate that cellular immunity is critical for the clearance ofB. microtiin BALB/c mice but that disease resolution can occur even in the absence of IFN-γ.
Identification of Genes Affecting
Salmonella enterica
Serovar Enteritidis Infection of Chicken Macrophages
Authors: Zhao Yixian , Jansen Ruud , Gaastra Wim , Arkesteijn Ger , van der Zeijst Bernard A. M. , van Putten Jos P. M.
Published: October 2002

Screening of 7,680
Salmonella enterica
serovar Enteritidis mutants for attenuation in a chicken macrophage infection model yielded a series of mutants including several with defects in previously unrecognized
Salmonella
virulence genes. One of the newly identified genes was the
pbpA2
gene, belonging to the penicillin binding protein gene family.
Identification of
Bacillus subtilis
CysL, a Regulator of the
cysJI
Operon, Which Encodes Sulfite Reductase
Authors: Guillouard Isabelle , Auger Sandrine , Hullo Marie-Françoise , Chetouani Farid , Danchin Antoine , Martin-Verstraete Isabelle
Published: October 2002

The way in which the genes involved in cysteine biosynthesis are regulated is poorly characterized in
Bacillus subtilis
. We showed that CysL (formerly YwfK), a LysR-type transcriptional regulator, activates the transcription of the
cysJI
operon, which encodes sulfite reductase. We demonstrated that a
cysL
mutant and a
cysJI
mutant have similar phenotypes. Both are unable to grow using sulfate or sulfite as the sulfur source. The level of expression of the
cysJI
operon is higher in the presence of sulfate, sulfite, or thiosulfate than in the presence of cysteine. Conversely, the transcription of the
cysH
and
cysK
genes is not regulated by these sulfur sources. In the presence of thiosulfate, the expression of the
cysJI
operon was reduced 11-fold, whereas the expression of the
cysH
and
cysK
genes was increased, in a
cysL
mutant. A
cis
-acting DNA sequence located upstream of the transcriptional start site of the
cysJI
operon (positions −76 to −70) was shown to be necessary for sulfur source- and CysL-dependent regulation. CysL also negatively regulates its own transcription, a common characteristic of the LysR-type regulators. Gel mobility shift assays and DNase I footprint experiments showed that the CysL protein specifically binds to
cysJ
and
cysL
promoter regions. This is the first report of a regulator of some of the genes involved in cysteine biosynthesis in
B. subtilis
.
Transposase-Dependent Formation of Circular IS256Derivatives inStaphylococcus epidermidisandStaphylococcus aureus
Authors: Loessner Isabel , Dietrich Katja , Dittrich Dorothea , Hacker Jörg , Ziebuhr Wilma
Published: October 2002

IS256is a highly active insertion sequence (IS) element of multiresistant staphylococci and enterococci. Here we show that, in aStaphylococcus epidermidisclinical isolate, as well as in recombinantStaphylococcus aureusandEscherichia colicarrying a single IS256insertion on a plasmid, IS256excises as an extrachromosomal circular DNA molecule. First, circles were identified that contained a complete copy of IS256. In this case, the sequence connecting the left and right ends of IS256was derived from flanking DNA sequences of the parental genetic locus. Second, circle junctions were detected in which one end of IS256was truncated. Nucleotide sequencing of circle junctions revealed that (i) either end of IS256can attack the opposite terminus and (ii) the circle junctions vary significantly in size. Upon deletion of the IS256open reading frame at the 3′ end and site-directed mutageneses of the putative DDE motif, circular IS256molecules were no longer detectable, which implicates the IS256-encoded transposase protein with the circularization of the element.
SoxRS Down-Regulation ofrobTranscription
Authors: Michán Carmen , Manchado Manuel , Pueyo Carmen
Published: October 2002

Rob is regarded as a constitutively expressed protein, although little is known about howrobgene is regulated. We show here by reverse transcription-PCR that the transcriptional levels ofrobare strongly down-regulated in response to the superoxide-generating agent paraquat (PQ). Repression reached a maximum of 20-fold after 10 min exposure at 10 μM PQ. The magnitude ofrobrepression was comparable to that of induction quantified for the most sensitive SoxS targets. β-Galactosidase expression with therob2::lacZtranscriptional fusion indicates that down-regulation ofrobexpression takes place, at least in part, at the level of transcription initiation. Moreover, ca. 50% of therobmRNA was degraded in <1 min after the addition of rifampin to inhibit transcription. This intrinsic short half-life, which is of obvious benefit for a rapid down-regulation after transcription ceases, was unaffected by the addition of PQ. No repression was observed in asoxR-null strain, indicating that therobtranscript level might be negatively modulated by the intracellular amounts of SoxS protein. Gel retardation assays support the idea that in vivo SoxS would blockrobtranscription directly.
A New Type IV Secretion System Promotes Conjugal Transfer inAgrobacterium tumefaciens
Authors: Chen Lishan , Chen Yuching , Wood Derek W. , Nester Eugene W.
Published: October 2002

Two DNA transfer systems encoded by the tumor-inducing (Ti) plasmid have been previously identified inAgrobacterium tumefaciens. ThevirBoperon is required for the transfer of transferred DNA to the plant host, and thetrbsystem encodes functions required for the conjugal transfer of the Ti plasmid between cells ofAgrobacterium. Recent availability of the genome sequence ofAgrobacteriumallowed us to identify a third system that is most similar to the VirB type IV secretion system ofBartonella henselae. We have designated this systemavhBforAgrobacteriumvirulence homologuevirB. TheavhBloci reside on pAtC58 and encode at least 10 proteins (AvhB2 through AvhB11), 7 of which display significant similarity to the corresponding virulence-associated VirB proteins of the Ti plasmid. However, the AvhB system is not required for tumor formation; rather, it mediates the conjugal transfer of the pAtC58 cryptic plasmid between cells ofAgrobacterium. This transfer occurs in the absence of the Ti plasmid-encoded VirB and Trb systems. Like the VirB system, AvhB products promote the conjugal transfer of the IncQ plasmid RSF1010, suggesting that these products comprise a mating-pair formation system. The presence of plasmid TiC58 or plasmid RSF1010 reduces the conjugal transfer efficiency of pAtC58 10- or 1,000-fold, respectively. These data suggest that complex substrate interactions exist among the three DNA transfer systems ofAgrobacterium.
N4 RNA Polymerase II, a Heterodimeric RNA Polymerase with Homology to the Single-Subunit Family of RNA Polymerases
Authors: Willis S. H. , Kazmierczak K. M. , Carter R. H. , Rothman-Denes L. B.
Published: October 2002

Bacteriophage N4 middle genes are transcribed by a phage-coded, heterodimeric, rifampin-resistant RNA polymerase, N4 RNA polymerase II (N4 RNAPII). Sequencing and transcriptional analysis revealed that the genes encoding the two subunits comprising N4 RNAPII are translated from a common transcript initiating at the N4 early promoter Pe3. These genes code for proteins of 269 and 404 amino acid residues with sequence similarity to the single-subunit, phage-like RNA polymerases. The genes encoding the N4 RNAPII subunits, as well as a synthetic construct encoding a fusion polypeptide, have been cloned and expressed. Both the individually expressed subunits and the fusion polypeptide reconstitute functional enzymes in vivo and in vitro.
prbA
, a Gene Coding for an Esterase Hydrolyzing Parabens in
Enterobacter cloacae
and
Enterobacter gergoviae
Strains
Authors: Valkova Nelly , Lépine François , Bollet Claude , Dupont Maryse , Villemur Richard
Published: October 2002

The new gene
prbA
encodes an esterase responsible for the hydrolysis of the ester bond of parabens in
Enterobacter cloacae
strain EM. This gene is located on the chromosome of strain EM and was cloned by several PCR approaches. The
prbA
gene codes for an immature protein of 533 amino acids, the first 31 of which represent a proposed signal peptide yielding a mature protein of a putative molecular mass of 54.6 kDa. This enzyme presents analogies with other type B carboxylesterases, mainly of eukaryotic origin. The cloning and expression of the
prbA
gene in a strain of
Escherichia coli
previously unable to hydrolyze parabens resulted in the acquisition of a hydrolytic capacity comparable to the original activity of strain EM, along with an increased resistance of the transformed strain to methyl paraben. The presence of homologues of
prbA
was tested in additional ubiquitous bacteria, which may be causative factors in opportunistic infections, including
Enterobacter gergoviae
,
Enterobacter aerogenes
,
Pseudomonas agglomerans
,
E. coli
,
Pseudomonas aeruginosa
, and
Burkholderia cepacia
. Among the 41 total strains tested, 2 strains of
E. gergoviae
and 1 strain of
Burkholderia cepacia
were able to degrade almost completely 800 mg of methyl paraben liter
−1
. Two strains of
E. gergoviae
, named G1 and G12, contained a gene that showed high homology to the
prbA
gene of
E. cloacae
and demonstrated comparable paraben esterase activities. The significant geographical distance between the locations of the isolated
E. cloacae
and
E. gergoviae
strains suggests the possibility of an efficient transfer mechanism of the
prbA
gene, conferring additional resistance to parabens in ubiquitous bacteria that represent a common source of opportunistic infections.
Transposition of Cyanobacterium Insertion Element ISY100inEscherichia coli
Authors: Urasaki Akihiro , Sekine Yasuhiko , Ohtsubo Eiichi
Published: October 2002

The genome of the cyanobacteriumSynechocystissp. strain PCC6803 has nine kinds of insertion sequence (IS) elements, of which ISY100in 22 copies is the most abundant. A typical ISY100member is 947 bp long and has imperfect terminal inverted repeat sequences. It has an open reading frame encoding a 282-amino-acid protein that appears to have partial homology with the transposase encoded by a bacterial IS, IS630, indicating that ISY100belongs to the IS630family. To determine whether ISY100has transposition ability, we constructed a plasmid carrying the IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible transposase gene at one site and mini-ISY100with the chloramphenicol resistance gene, substituted for the transposase gene of ISY100, at another site and introduced the plasmid into anEscherichia colistrain already harboring a target plasmid. Mini-ISY100transposed to the target plasmid in the presence of IPTG at a very high frequency. Mini-ISY100was inserted into the TA sequence and duplicated it upon transposition, as do IS630family elements. Moreover, the mini-ISY100-carrying plasmid produced linear molecules of mini-ISY100with the exact 3′ ends of ISY100and 5′ ends lacking two nucleotides of the ISY100sequence. No bacterial insertion elements have been shown to generate such molecules, whereas the eukaryotic Tc1/marinerfamily elements, Tc1and Tc3, which transpose to the TA sequence, have. These findings suggest that ISY100transposes to a new site through the formation of linear molecules, such as Tc1and Tc3, by excision. Some Tc1/marinerfamily elements leave a footprint with an extra sequence at the site of excision. No footprints, however, were detected in the case of ISY100, suggesting that eukaryotes have a system that repairs a double strand break at the site of excision by an end-joining reaction, in which the gap is filled with a sequence of several base pairs, whereas prokaryotes do not have such a system. ISY100transposes inE. coli, indicating that it transposes without any host factor other than the transposase encoded by itself. Therefore, it may be able to transpose in other biological systems.
Multicenter Comparative Evaluation of Six Commercial Systems and the National Committee for Clinical Laboratory Standards M27-A Broth Microdilution Method for Fluconazole Susceptibility Testing of
Candida
Species
Authors: Morace G. , Amato G. , Bistoni F. , Fadda G. , Marone P. , Montagna M. T. , Oliveri S. , Polonelli L. , Rigoli R. , Mancuso I. , La Face S. , Masucci L. , Romano L. , Napoli C. , Tatò D. , Buscema M. G. , Belli C. M. C. , Piccirillo M. M. , Conti S. , Covan S. , Fanti F. , Cavanna C. , D'Alò F. , Pitzurra L.
Published: October 2002

Fluconazole susceptibility among 800 clinical
Candida
isolates (60%
C. albicans
) and two control strains (
C. krusei
ATCC 6258 and
C. parapsilosis
ATCC 22019) was tested with the NCCLS M27-A method (gold standard) and six commercial products (Candifast, disk, Etest, Fungitest, Integral System Yeasts, and Sensititre YeastOne). Results were classified as susceptible, susceptible-dose dependent, or resistant using M27-A breakpoints or, for Fungitest, Integral System Yeasts, and Candifast, as susceptible, intermediate, or resistant, according to the manufacturers' instructions. Concordance with NCCLS M27-A results was analyzed with the χ
2
test. Intra- and interlaboratory reproducibility was also evaluated. NCCLS M27-A (90.1%), Etest (93.1%), Sensititre YeastOne (93.1%), disk (96.7%), Fungitest (92.6%), Integral System Yeasts (40.6%), and Candifast (6.0%) classified the indicated percentages of
C. albicans
isolates as susceptible. Among non-
C. albicans
strains, the percentages of susceptible isolates were as follows: NCCLS M27-A, 74.0%; Etest, 83.8%; Sensititre YeastOne, 64.1%; disk, 60.6%; Fungitest, 76.6%; Integral System Yeasts, 28.3%; and Candifast, 27.4%. All methods except Candifast and Integral System Yeasts showed good agreement with NCCLS M27-A results for both
C albicans
and non-
C. albicans
isolates. Intralaboratory reproducibility was excellent for NCCLS M27-A, Etest, Sensititre YeastOne, disk, and Fungitest (88 to 91%). Similar results emerged from the interlaboratory reproducibility evaluation. Our findings indicate that some commercial methods can be useful for fluconazole susceptibility testing of clinical
Candida
isolates. Those characterized by a lack of medium standardization and/or objective interpretative criteria should be avoided. Particular caution is necessary when testing is being done for clinical and epidemiological purposes.
Simple and Rapid Differentiation ofMycobacterium tuberculosisH37Ra fromM. tuberculosisClinical Isolates through Two Cytochemical Tests Using Neutral Red and Nile Blue Stains
Authors: Soto Carlos Y. , Andreu Núria , Gibert Isidre , Luquin Marina
Published: October 2002

The attenuatedMycobacterium tuberculosisstrain H37Ra is one of the most commonly used controls forM. tuberculosisidentification in the clinical laboratory and is a source of false-positive results forM. tuberculosisas a consequence of cross-contamination. Therefore, the ability to discriminate between H37Ra and real clinical isolates has important public health implications. To date, differentiation of H37Ra fromM. tuberculosisclinical isolates is possible only by IS6110genotyping and spoligotyping. In the 1950s, some authors reported that the virulent strain H37Rv andM. tuberculosisclinical isolates were able to fix basic dyes in their anionic forms, while H37Ra was not. We have studied the different techniques described forM. tuberculosiscytochemical staining and have chosen the best of these, introducing certain modifications in order to increase their discriminative power and reproducibility. We describe cytochemical staining ofM. tuberculosiscells with neutral red and Nile blue, which differentiates H37Ra from virulent strains. This method could be used as an easy laboratory tool for distinguishing between H37Ra and realM. tuberculosisclinical isolates.
Assessment of Mycobacterial, Propionibacterial, and Human Herpesvirus 8 DNA in Tissues of Greek Patients with Sarcoidosis
Authors: Gazouli M. , Ikonomopoulos J. , Trigidou R. , Foteinou M. , Kittas C. , Gorgoulis V.
Published: October 2002

The causes of sarcoidosis are unknown. In this study, we report the presence of
Mycobacterium tuberculosis
complex and
Propionibacterium granulosum
DNA in a significant proportion of Greek patients with sarcoidosis. Human herpesvirus 8 DNA was not detected in sarcoid tissues from Greek patients. Our findings are discussed.
Effects of Repeated Subculturing and Prolonged Storage at Room Temperature of Enterohemorrhagic
Escherichia coli
O157:H7 on Pulsed-Field Gel Electrophoresis Profiles
Authors: Iguchi Atsushi , Osawa Ro , Kawano Junichi , Shimizu Akira , Terajima Jun , Watanabe Haruo
Published: October 2002

Three clinical strains of enterohemorrhagic
Escherichia coli
O157:H7 which were subcultured repeatedly or stored at room temperature over a 25-week period showed appreciable variations in their pulsed-field gel electrophoresis fragment patterns. The variations could be explained by a couple of spontaneous genetic events at most and thus did not invalidate the genetic lineage of the strains.
Identification and Expression of Human Cytomegalovirus Transcription Units Coding for Two Distinct Fcγ Receptor Homologs
Authors: Atalay Ramazan , Zimmermann Albert , Wagner Markus , Borst Eva , Benz Christine , Messerle Martin , Hengel Hartmut
Published: October 2002

Cellular receptors for the Fc domain of immunoglobulin G (IgG) (FcγRs) comprise a family of surface receptors on immune cells connecting humoral and cellular immune responses. Several herpesviruses induce FcγR activities in infected cells. Here we identify two distinct human cytomegalovirus (HCMV)-encoded vFcγR glycoproteins of 34 and 68 kDa. A panel of HCMV strains exhibited a slight molecular microheterogeneity between Fcγ-binding proteins, suggesting their viral origin. To locate the responsible genes within the HCMV genome, a large set of targeted HCMV deletion mutants was constructed. The mutant analysis allowed the identification of a splicedUL119-UL118mRNA to encode vFcγR gp68 andTRL11/IRL11to encode vFcγR gp34. Both vFcγRs are surface resident type I transmembrane glycoproteins. Significant relatedness of sequences in the extracellular chain of gpUL119-118 and gpTRL11 with particular immunoglobulin supergene family domains present in FcγR I and FcγRs II/III, respectively, indicates a different ancestry and function of gpUL119-118 and gpTRL11. The HCMV-encoded vFcγRs highlight an impressive diversification and redundancy of FcγR structures.
Human Cytomegalovirus UL84 Localizes to the Cell Nucleus via a Nuclear Localization Signal and Is a Component of Viral Replication Compartments
Authors: Xu Yiyang , Colletti Kelly S. , Pari Gregory S.
Published: October 2002

The UL84 open reading frame encodes a protein that is required for origin-dependent DNA replication and interacts with the immediate-early protein IE2 in lytically infected cells. Transfection of UL84 expression constructs showed that UL84 localized to the nucleus of transfected cells in the absence of any other viral proteins and displayed a punctate speckled fluorescent staining pattern. Cotransfection of all the human cytomegalovirus replication proteins and
ori
Lyt, along with pUL84-EGFP, showed that UL84 colocalized with UL44 (polymerase accessory protein) in replication compartments. Experiments using infected human fibroblasts demonstrated that UL84 also colocalized with UL44 and IE2 in viral replication compartments in infected cells. A nuclear localization signal was identified using plasmid constructs expressing truncation mutants of the UL84 protein in transient transfection assays. Transfection assays showed that UL84 failed to localize to the nucleus when 200 amino acids of the N terminus were deleted. Inspection of the UL84 amino acid sequence revealed a consensus putative nuclear localization signal between amino acids 160 and 171 (PEKKKEKQEKK) of the UL84 protein.
Genetic Diversity within Human Erythroviruses: Identification of Three Genotypes
Authors: Servant Annabelle , Laperche Syria , Lallemand Francis , Marinho Valérie , De Saint Maur Guillemette , Meritet Jean François , Garbarg-Chenon Antoine
Published: October 2002

B19 virus is a human virus belonging to the genus
Erythrovirus
. The genetic diversity among B19 virus isolates has been reported to be very low, with less than 2% nucleotide divergence in the whole genome sequence. We have previously reported the isolation of a human erythrovirus isolate, termed V9, whose sequence was markedly distinct (>11% nucleotide divergence) from that of B19 virus. To date, the V9 isolate remains the unique representative of a new variant in the genus
Erythrovirus
, and its taxonomic position is unclear. We report here the isolation of 11 V9-related viruses. A prospective study conducted in France between 1999 and 2001 indicates that V9-related viruses actually circulate at a significant frequency (11.4%) along with B19 viruses. Analysis of the nearly full-length genome sequence of one V9-related isolate (D91.1) indicates that the D91.1 sequence clusters together with but is notably distant from the V9 sequence (5.3% divergence) and is distantly related to B19 virus sequences (13.8 to 14.2% divergence). Additional phylogenetic analysis of partial sequences from the V9-related isolates combined with erythrovirus sequences available in GenBank indicates that the erythrovirus group is more diverse than thought previously and can be divided into three well-individualized genotypes, with B19 viruses corresponding to genotype 1 and V9-related viruses being distributed into genotypes 2 and 3.