bluuebunny/crossref_metadata_2025 huggingface dataset:
High-Efficiency System for the Construction of Adenovirus Vectors and Its Application to the Generation of Representative Adenovirus-Based cDNA Expression Libraries
Authors: Hillgenberg Moritz , Hofmann Christian , Stadler Herbert , Löser Peter
Published: May 2006

We here describe a convenient system for the production of recombinant
adenovirus vectors and its use for the construction of a representative
adenovirus-based cDNA expression library. The system is based on direct
site-specific insertion of transgene cassettes into a replicating donor
virus. The transgene is inserted into a donor plasmid containing the
viral 5′ inverted terminal repeat, the complete viral packaging
signal, and a single
loxP
site. The plasmid is then
transfected into a Cre recombinase-expressing packaging cell line that
has been infected with a donor virus containing a partially deleted
packaging signal flanked by
loxP
sites. Cre recombinase, by
two steps of action, sequentially catalyzes the generation of a
nonpackageable donor virus acceptor substrate and the generation of the
desired recombinant adenovirus vector. Due to its growth impairment,
residual donor virus can efficiently be counterselected during
amplification of the recombinant adenovirus vector. By using this
adenovirus construction system, a plasmid-based human liver cDNA
library was converted by a single step into an adenovirus-based cDNA
expression library with about 10
6
independent adenovirus
clones. The high-titer purified library was shown to contain about 44%
of full-length cDNAs with an average insert size of 1.3 kb. cDNAs of a
gene expressed at a high level (human α
1
-antitrypsin) and a gene expressed at a relatively
low level (human coagulation factor IX) in human liver were isolated
from the adenovirus-based library using an enzyme-linked immunosorbent
assay-based screening procedure.
Gag Regulates Association of Human Immunodeficiency Virus Type 1 Envelope with Detergent-Resistant Membranes
Authors: Bhattacharya Jayanta , Repik Alexander , Clapham Paul R.
Published: May 2006

Assembly of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein on budding virus particles is important for efficient infection of target cells. In infected cells, lipid rafts have been proposed to form platforms for virus assembly and budding. Gag precursors partly associate with detergent-resistant membranes (DRMs) that are believed to represent lipid rafts. The cytoplasmic domain of the envelope gp41 usually carries palmitate groups that were also reported to confer DRM association. Gag precursors confer budding and carry envelope glycoproteins onto virions via specific Gag-envelope interactions. Thus, specific mutations in both the matrix domain of the Gag precursor and gp41 cytoplasmic domain abrogate envelope incorporation onto virions. Here, we show that HIV-1 envelope association with DRMs is directly influenced by its interaction with Gag. Thus, in the absence of Gag, envelope fails to associate with DRMs. A mutation in the p17 matrix (L30E) domain in Gag (Gag L30E) that abrogates envelope incorporation onto virions also eliminated envelope association with DRMs in 293T cells and in the T-cell line, MOLT 4. These observations are consistent with a requirement for an Env-Gag interaction for raft association and subsequent assembly onto virions. In addition to this observation, we found that mutations in the gp41 cytoplasmic domain that abrogated envelope incorporation onto virions and impaired infectivity of cell-free virus also eliminated envelope association with DRMs. On the basis of these observations, we propose that Gag-envelope interaction is essential for efficient envelope association with DRMs, which in turn is essential for envelope budding and assembly onto virus particles.
Inhibition of DNA Replication of Human Papillomavirus by Artificial Zinc Finger Proteins
Authors: Mino Takashi , Hatono Takeaki , Matsumoto Naoki , Mori Tomoaki , Mineta Yusuke , Aoyama Yasuhiro , Sera Takashi
Published: May 2006

Recently,
we demonstrated that plant DNA virus replication was inhibited in
planta by using an artificial zinc finger protein (AZP) and created
AZP-based transgenic plants resistant to DNA virus infection. Here we
apply the AZP technology to the inhibition of replication of a
mammalian DNA virus, human papillomavirus type 18 (HPV-18). Two AZPs,
designated AZP
HPV
-1 and AZP
HPV
-2, were designed
by using our nondegenerate recognition code table and were constructed
to block binding of the HPV-18 E2 replication protein to the
replication origin. Both of the newly designed AZPs had much higher
affinities towards the replication origin than did the E2 protein, and
they efficiently blocked E2 binding in vitro. In transient replication
assays, both AZPs inhibited viral DNA replication, especially
AZP
HPV
-2, which reduced the replication level to
approximately 10%. We also demonstrated in transient replication
assays, using plasmids with mutant replication origins, that
AZP
HPV
-2 could precisely recognize the replication origin in
mammalian cells. Thus, it was demonstrated that the AZP technology
could be applied not only to plant DNA viruses but also to mammalian
DNA
viruses.
Inhibition of the NF-κB Pathway by Varicella-Zoster Virus In Vitro and in Human Epidermal Cells In Vivo
Authors: Jones Jeremy O. , Arvin Ann M.
Published: May 2006

Varicella-zoster virus (VZV) is an alphaherpesvirus that causes varicella and herpes zoster. Using human cellular DNA microarrays, we found that many nuclear factor kappa B (NF-κB)-responsive genes were down-regulated in VZV-infected fibroblasts, suggesting that VZV infection inhibited the NF-κB pathway. The activation of this pathway causes a cellular antiviral response, including the production of alpha/beta interferon, cytokines, and other proteins that restrict viral infection. In these experiments, we demonstrated that VZV interferes with NF-κB activation in cultured fibroblasts and in differentiated epidermal cells in skin xenografts of SCIDhu mice infected in vivo. VZV infection of fibroblasts caused a transient nuclear translocation of p50 and p65, the canonical NF-κB family members. In a process that was dependent upon the presence of infectious VZV, these proteins rapidly became sequestered in the cytoplasm of VZV-infected cells. Exclusion of NF-κB proteins from nuclei was associated with the continued presence of IκBα, which binds p50 and p65 and prevents their nuclear accumulation. IκBα levels did not diminish even though the protein became phosphorylated and ubiquitinated, as determined based on detection of the characteristic high-molecular-weight form of the protein, and the 26S proteasome remained functional in VZV-infected cells. VZV infection also inhibited the characteristic degradation of IκBα that is induced by exposure of fibroblasts to tumor necrosis factor alpha. As expected, herpes simplex virus 1 caused the persistent nuclear translocation of NF-κB proteins, which has been shown to facilitate its replication, whereas VZV infection progressed without persistent NF-κB nuclear localization. We suggest that VZV has evolved a mechanism to limit host cell antiviral defenses by sequestering NF-κB proteins in the cytoplasm, a strategy that appears to be unique among the herpesviruses.
Nuclear Localization of Flavivirus RNA Synthesis in Infected Cells
Authors: Uchil Pradeep Devappa , Kumar Anil V. A. , Satchidanandam Vijaya
Published: May 2006

Flaviviral replication is believed to be exclusively cytoplasmic, occurring within virus-induced membrane-bound replication complexes in the host cytoplasm. Here we show that a significant proportion (20%) of the total RNA-dependent RNA polymerase (RdRp) activity from cells infected with West Nile virus, Japanese encephalitis virus (JEV), and dengue virus is resident within the nucleus. Consistent with this, the major replicase proteins NS3 and NS5 of JEV also localized within the nucleus. NS5 was found distributed throughout the nucleoplasm, but NS3 was present at sites of active flaviviral RNA synthesis, colocalizing with NS5, and visible as distinct foci along the inner periphery of the nucleus by confocal and immunoelectron microscopy. Both these viral replicase proteins were also present in the nuclear matrix, colocalizing with the peripheral lamina, and revealed a well-entrenched nuclear location for the viral replication complex. In keeping with this observation, antibodies to either NS3 or NS5 coimmunoprecipitated the other protein from isolated nuclei along with newly synthesized viral RNA. Taken together these data suggest an absolute requirement for both of the replicase proteins for nucleus-localized synthesis of flavivirus RNA. Thus, we conclusively demonstrate for the first time that the host cell nucleus functions as an additional site for the presence of functionally active flaviviral replicase complex.
Mucosal Administration of CpG Oligodeoxynucleotide Elicits Strong CC and CXC Chemokine Responses in the Vagina and Serves as a Potent Th1-Tilting Adjuvant for Recombinant gD2 Protein Vaccination against Genital Herpes
Authors: Tengvall Sara , Lundqvist Annika , Eisenberg Roselyn J. , Cohen Gary H. , Harandi Ali M.
Published: May 2006

Although sexually transmitted pathogens are capable of inducing pathogen-specific immune responses, vaginal administration of nonreplicating antigens elicits only weak, nondisseminating immune responses. The present study was undertaken to examine the potential of CpG-containing oligodeoxynucleotide (CpG ODN) for induction of chemokine responses in the genital tract mucosa and also as a vaginal adjuvant in combination with glycoprotein D of herpes simplex virus type 2 (HSV-2) for induction of antigen-specific immune responses. We found that a single intravaginal administration of CpG ODN in mice stimulates a rapid and potent response of CC chemokines macrophage inflammatory protein 1α (MIP-1α), MIP-1β, and RANTES as well as of CXC chemokines MIP-2 and IP-10 in the vagina and/or the genital lymph nodes. Importantly, intravaginal vaccination with recombinant gD2 in combination with CpG ODN gave rise to a strong antigen-specific Th1-like immune response in the genital lymph nodes as well as the spleens of the vaccinated mice. Further, such an immunization scheme conferred both systemic and mucosal immunoglobulin G antibody responses as well as protection against an otherwise lethal vaginal challenge with HSV-2. These results illustrate the potential of CpG ODN for induction of potent chemokine responses in the genital tract and also as a vaginal adjuvant for generation of Th1-type mucosal and systemic immune responses towards a nonreplicating antigen derived from a sexually transmitted pathogen. These data have implications for the development of a mucosal vaccine against genital herpes and possibly other sexually transmitted diseases.
Ebola Virus VP35 Protein Binds Double-Stranded RNA and Inhibits Alpha/Beta Interferon Production Induced by RIG-I Signaling
Authors: Cárdenas Washington B. , Loo Yueh-Ming , Gale Michael , Hartman Amy L. , Kimberlin Christopher R. , Martínez-Sobrido Luis , Saphire Erica Ollmann , Basler Christopher F.
Published: May 2006

The Ebola virus (EBOV) VP35 protein blocks the virus-induced phosphorylation and activation of interferon regulatory factor 3 (IRF-3), a transcription factor critical for the induction of alpha/beta interferon (IFN-α/β) expression. However, the mechanism(s) by which this blockage occurs remains incompletely defined. We now provide evidence that VP35 possesses double-stranded RNA (dsRNA)-binding activity. Specifically, VP35 bound to poly(rI) · poly(rC)-coated Sepharose beads but not control beads. In contrast, two VP35 point mutants, R312A and K309A, were found to be greatly impaired in their dsRNA-binding activity. Competition assays showed that VP35 interacted specifically with poly(rI) · poly(rC), poly(rA) · poly(rU), or in vitro-transcribed dsRNAs derived from EBOV sequences, and not with single-stranded RNAs (ssRNAs) or double-stranded DNA. We then screened wild-type and mutant VP35s for their ability to target different components of the signaling pathways that activate IRF-3. These experiments indicate that VP35 blocks activation of IRF-3 induced by overexpression of RIG-I, a cellular helicase recently implicated in the activation of IRF-3 by either virus or dsRNA. Interestingly, the VP35 mutants impaired for dsRNA binding have a decreased but measurable IFN antagonist activity in these assays. Additionally, wild-type and dsRNA-binding-mutant VP35s were found to have equivalent abilities to inhibit activation of the IFN-β promoter induced by overexpression of IPS-1, a recently identified signaling molecule downstream of RIG-I, or by overexpression of the IRF-3 kinases IKKε and TBK-1. These data support the hypothesis that dsRNA binding may contribute to VP35 IFN antagonist function. However, additional mechanisms of inhibition, at a point proximal to the IRF-3 kinases, most likely also exist.
A Short Isoform of Human Cytomegalovirus US3 Functions as a Dominant Negative Inhibitor of the Full-Length Form
Authors: Shin Jinwook , Park Boyoun , Lee Sungwook , Kim Youngkyun , Biegalke Bonita J. , Kang Seongman , Ahn Kwangseog
Published: May 2006

Human cytomegalovirus encodes four unique short (US) region proteins, each of which is independently sufficient for causing the down-regulation of major histocompatibility complex (MHC) class I molecules on the cell surface. This down-regulation enables infected cells to evade recognition by cytotoxic T lymphocytes (CTLs) but makes them vulnerable to lysis by natural killer (NK) cells, which lyse those cells that lack MHC class I molecules. The 22-kDa US3 glycoprotein is able to down-regulate the surface expression of MHC class I molecules by dual mechanisms: direct endoplasmic reticulum retention by physical association and/or tapasin inhibition. The alternative splicing of theUS3gene generates two additional products, including 17-kDa and 3.5-kDa truncated isoforms; however, the functional significance of these isoforms during viral infection is unknown. Here, we describe a novel mode of self-regulation of US3 function that uses the endogenously produced truncated isoform. The truncated isoform itself neither binds to MHC class I molecules nor prevents the full-length US3 from interacting with MHC class I molecules. Instead, the truncated isoform associates with tapasin and competes with full-length US3 for binding to tapasin; thus, it suppresses the action of US3 that causes the disruption of the function of tapasin. Our results indicate that the truncated isoform of theUS3locus acts as a dominant negative regulator of full-length US3 activity. These data reflect the manner in which the virus has developed temporal survival strategies during viral infection against immune surveillance involving both CTLs and NK cells.
Batai and Ngari Viruses: M Segment Reassortment and Association with Severe Febrile Disease Outbreaks in East Africa
Authors: Briese Thomas , Bird Brian , Kapoor Vishal , Nichol Stuart T. , Lipkin W. Ian
Published: May 2006

Ngari virus is an orthobunyavirus recently recognized as a reassortant between Bunyamwera virus and an as yet unidentified M segment donor. Analysis of M segment sequences of Batai and Ilesha viruses revealed 95% deduced amino acid identity between Batai virus and Ngari virus. These findings suggest Batai virus as the donor of Ngari virus M segment sequence. Analysis of Batai virus-related African isolates identified UgMP-6830, isolated from mosquitoes in Uganda, as an isolate of Batai virus. KV-141, isolated during a febrile disease outbreak in Sudan, was identified as another isolate of Ngari virus, emphasizing a role of this reassortant virus in severe human illness throughout East Africa.
Acetylation of the Latency-Associated Nuclear Antigen Regulates Repression of Kaposi's Sarcoma-Associated Herpesvirus Lytic Transcription
Authors: Lu Fang , Day Latasha , Gao S.-J. , Lieberman Paul M.
Published: May 2006

Reactivation of the Kaposi's sarcoma-associated herpesvirus (KSHV) lytic cycle can be initiated by transcription activation of the ORF50 immediate early gene (Rta). We show that ORF50 transcription is actively repressed by the KSHV latency-associated nuclear antigen (LANA) during latency. Depletion of LANA by small interfering RNA derepressed ORF50 transcription in the latently infected BCBL1 pleural effusion lymphoma-derived cell line. In contrast, overexpression of LANA suppressed ORF50 mRNA levels in BCBL1 cells. ORF50 transcription was significantly elevated during primary infection with recombinant virus lacking LANA, further indicating that LANA plays a role in lytic gene silencing during the establishment of latency. Chromatin immunoprecipitation assays indicated that LANA interacts with the ORF50 promoter region in latently infected cells. Histone deacetylase inhibitors, including sodium butyrate (NaB) and trichostatin A, caused the rapid dissociation of LANA from the ORF50 promoter. NaB treatment of latently infected BCBL1 cells disrupted a stable interaction between LANA and the cellular proteins Sp1 and histone H2B. We also found immunological and radiochemical evidence that LANA is subject to lysine acetylation after NaB treatment. These findings support the role of LANA as a transcriptional repressor of lytic reactivation and provide evidence that lysine acetylation regulates LANA interactions with chromatin, Sp1, and ORF50 promoter DNA.
Transspecies Transmission of the Endogenous Koala Retrovirus
Authors: Fiebig Uwe , Hartmann Manuel Garcia , Bannert Norbert , Kurth Reinhard , Denner Joachim
Published: May 2006

The koala retrovirus (KoRV) is a gammaretrovirus closely related to the gibbon ape leukemia virus and induces leukemias and immune deficiencies associated with opportunistic infections, such as chlamydiosis. Here we characterize a KoRV newly isolated from an animal in a German zoo and show infection of human and rat cell lines in vitro and of rats in vivo, using immunological and PCR methods for virus detection. The KoRV transmembrane envelope protein (p15E) was cloned and expressed, and p15E-specific neutralizing antibodies able to prevent virus infection in vitro were developed. Finally, evidence for immunosuppressive properties of the KoRV was obtained.
A Herpes Simplex Virus Recombinant That Exhibits a Single-Chain Antibody to HER2/neu Enters Cells through the Mammary Tumor Receptor, Independently of the gD Receptors
Authors: Menotti Laura , Cerretani Arianna , Campadelli-Fiume Gabriella
Published: May 2006

The human epidermal growth factor receptor 2/neuregulin (HER2/neu) receptor is overexpressed in highly malignant mammary and ovarian tumors and correlates with a poor prognosis. It is a target for therapy; humanized monoclonal antibodies to HER2 have led to increased survival of patients with HER2/neu-positive breast cancer. As a first step in the design of an oncolytic herpes simplex virus able to selectively infect HER2/neu-positive cells, we constructed two recombinants, R-LM11 and R-LM11L, that carry a single-chain antibody (scFv) against HER2 inserted at residue 24 of gD. The inserts were 247 or 256 amino acids long, and the size of the gD ectodomain was almost doubled by the insertion. We report the following. R-LM11 and R-LM11L infected derivatives of receptor-negative J or CHO cells that expressed HER2/neu as the sole receptor. Entry was dependent on HER2/neu, since it was inhibited in a dose-dependent manner by monoclonal antibodies to HER2/neu and by a soluble form of the receptor. The scFv insertion in gD disrupted the ability of the virus to enter cells through HVEM but maintained the ability to enter through nectin1. This report provides proof of principle that gD can tolerate fusion to a heterologous protein almost as large as the gD ectodomain itself without loss of profusion activity. Because the number of scFv's to a variety of receptors is continually increasing, this report makes possible the specific targeting of herpes simplex virus to a large collection of cell surface molecules for both oncolytic activity and visualization of tumor cells.
Application of comparative genomics in developing molecular markers tightly linked to the virus resistance geneRsv4 in soybean
Authors: Hwang Tae-Young , Moon Jung-Kyung , Yu Seok , Yang Kiwoung , Mohankumar Subbarayalu , Yu Yong Hwan , Lee Yeong Ho , Kim Hong Sig , Kim Hwan Mook , Maroof M A. Saghai , Jeong Soon-Chun
Published: May 2006

The Rsv4 gene confers resistance to all the known strain groups of soybean mosaic virus in soybean (Glycine max (L.) Merr.). To construct a fine genetic map near Rsv4 in soybean, we employed a comparative genomics approach that used genome sequence information of the model legume Lotus japonicus. Sequences of the soybean expressed sequence tags (ESTs) AI856415 and BF070293 mapping to one side of the Rsv4 gene showed high similarity with gene sequences of the transformation-competent artificial chromosome (TAC) clone LjT32P24 of Lotus. LjT32P24 is tightly linked to another sequenced TAC clone, LjT26I01, in Lotus. A new marker, AW307114A, developed from soybean EST AW307114, which is homologous to a Lotus gene within LjT26I01, was mapped to the other side of the Rsv4 gene. The identification of the microsyntenic relationship facilitated the development of additional 2 EST markers between BF070293-S and AW307114A bracketing the Rsv4 gene. Several other markers developed in this study were mapped to putative homoeologous or duplicated chromosomal regions in soybean. Alignment between the soybean maps indicated that Rsv4 is located near a local chromosomal rearrangement. This targeted comparative mapping serves to provide a foundation for marker-assisted selection and cloning of the Rsv4 gene.Key words: comparative genomics, resistance gene, lotus, soybean.
Karyological analysis of an interspecific hybrid between the dioeciousSilene latifoliaand the hermaphroditicSilene viscosa
Authors: Markova Michaela , Lengerova Martina , Zluvova Jitka , Janousek Bohuslav , Vyskot Boris
Published: May 2006

The genus Silene is a good model for studying evolution of the sex chromosomes, since it includes species that are hermaphroditic and dioecious, while maintain a basic chromosome number of 2n = 24. For some combinations of Silene species it is possible to construct interspecific hybrids. Here, we present a detailed karyological analysis of a hybrid between the dioecious Silene latifolia as the maternal plant and a related species, hermaphroditic Silene viscosa, used as a pollen partner. Using genomic probes (the genomic in situ hybridization (GISH) technique), we were able to clearly discriminate parental genomes and to show that they are largely separated in distinct nuclear domains. Molecular GISH and fluorescence in situ hybridization (FISH) markers document that the hybrid genome of somatic cells was strictly additive and stable, and that it had 12 chromosomes originating from each parent, including the only X chromosome of S. latifolia. Meiotic analysis revealed that, although related, respective parental chromosomes did not pair or paired only partially, which resulted in frequent chromosome abnormalities such as bridges and irregular non-disjunctions. GISH and FISH markers clearly document that the larger genome of S. latifolia and its largest chromosome component, the X chromosome, were mostly employed in chromosome lagging and misdivision.Key words: sex chromosome, Silene, interspecific hybrid, meiotic pairing, misdivision.
Analysis of ESTs from multiple Gossypium hirsutum tissues and identification of SSRs
Authors: Taliercio Earl , Allen Randy D , Essenberg Margaret , Klueva Natalya , Nguyen Henry , Patil Mohini A , Payton Paxton , Millena Ana Cecilia M. , Phillips Angela L , Pierce Margaret L , Scheffler Brian , Turley Rickie , Wang Jing , Zhang Deshui , Scheffler Jodi
Published: May 2006

In an effort to expand the Gossypium hirsutum L. (cotton) expressed sequence tag (EST) database, ESTs representing a variety of tissues and treatments were sequenced. Assembly of these sequences with ESTs already in the EST database (dbEST, GenBank) identified 9675 cotton sequences not present in GenBank. Statistical analysis of a subset of these ESTs identified genes likely differentially expressed in stems, cotyledons, and drought-stressed tissues. Annotation of the differentially expressed cDNAs tentatively identified genes involved in lignin metabolism, starch biosynthesis and stress response, consistent with pathways likely to be active in the tissues under investigation. Simple sequence repeats (SSRs) were identified among these ESTs, and an inexpensive method was developed to screen genomic DNA for the presence of these SSRs. At least 69 SSRs potentially useful in mapping were identified. Selected amplified SSRs were isolated and sequenced. The sequences corresponded to the EST containing the SSRs, confirming that these SSRs will potentially map the gene represented by the EST. The ESTs containing SSRs were annotated to help identify the genes that may be mapped using these markers.Key words: drought stress, gene annotation, gene mapping, tentative consensus sequence (TC), Xanthomonas campestris.
Farnesol restores wild-type colony morphology to 96% ofCandida albicanscolony morphology variants recovered following treatment with mutagens
Authors: Jensen Ellen C , Hornby Jacob M , Pagliaccetti Nicole E , Wolter Chuleeon M , Nickerson Kenneth W , Atkin Audrey L
Published: May 2006

Candida albicans is a diploid fungus that undergoes a morphological transition between budding yeast, hyphal, and pseudohyphal forms. The morphological transition is strongly correlated with virulence and is regulated in part by quorum sensing. Candida albicans produces and secretes farnesol that regulates the yeast to mycelia morphological transition. Mutants that fail to synthesize or respond to farnesol could be locked in the filamentous mode. To test this hypothesis, a collection of C. albicans mutants were isolated that have altered colony morphologies indicative of the presence of hyphal cells under environmental conditions where C. albicans normally grows only as yeasts. All mutants were characterized for their ability to respond to farnesol. Of these, 95.9% fully or partially reverted to wild-type morphology on yeast malt (YM) agar plates supplemented with farnesol. All mutants that respond to farnesol regained their hyphal morphology when restreaked on YM plates without farnesol. The observation that farnesol remedial mutants are so common (95.9%) relative to mutants that fail to respond to farnesol (4.1%) suggests that farnesol activates and (or) induces a pathway that can override many of the morphogenesis defects in these mutants. Additionally, 9 mutants chosen at random were screened for farnesol production. Two mutants failed to produce detectable levels of farnesol.Key words: farnesol-remedial mutants, farnesol-sensing mutants, farnesol-synthesis mutants, quorum sensing, Candida albicans, morphological transition.
Newly featured infection events in a supernodulating soybean mutant SS2-2 by Bradyrhizobium japonicum
Authors: Lestari Puji , Van Kyujung , Kim Moon Young , Lee Byun-Woo , Lee Suk-Ha
Published: May 2006

Supernodulating soybean (Glycine max L. Merr.) mutant SS2-2 and its wild-type counterpart, Sinpaldalkong 2, were examined for the microstructural events associated with nodule formation and development. SS2-2 produced a substantially higher percentage of curled root hairs than the wild type, especially at 14 days after inoculation with Bradyrhizobium japonicum. In addition, there was new evidence that in SS2-2, B. japonicum also entered through fissures created by the emerging adventitious root primordia. Early steps of nodule ontogeny were faster in SS2-2, and continued development of initiated nodules was more frequent and occurred at a higher frequency than in the wild type. These data suggest that the early expression of autoregulation is facilitated by decreasing the speed of cortical cell development, leading to the subsequent termination of less-developed nodules. The nodules of SS2-2 developed into spherical nodules like those formed on the wild type. In both the wild type and supernodulating mutant, vascular bundles bifurcate from root stele and branch off in the nodule cortex to surround the central infected zone. These findings indicate that SS2-2 has complete endosymbiosis and forms completely developed nodule vascular bundles like the wild type, but that the speed of nodule ontogeny differs between the wild type and SS2-2. Thus, SS2-2 has a novel symbiotic phenotype with regard to nodule organogenesis.Key words: Bradyrhizobium japonicum, early nodule development, Glycine max, root hair curling, supernodulation.
Effects of amoebae on the growth of microbes isolated from moisture-damaged buildings
Authors: Yli-Pirilä Terhi , Kusnetsov Jaana , Hirvonen Maija-Riitta , Seuri Markku , Nevalainen Aino
Published: May 2006

Dampness, moisture, and mold in buildings are associated with adverse health outcomes. In addition to fungi and bacteria, amoebae have been found in moisture-damaged building materials. Amoebae and a growing list of bacteria have been shown to have mutual effects on each other's growth, but the interactions between amoebae and microbes common in moisture-damaged buildings have not been reported. We co-cultivated the amoeba Acanthamoeba polyphaga with bacteria and fungi isolated from moisture-damaged buildings in laboratory conditions for up to 28 days. The microbes selected were the bacteria Streptomyces californicus, Bacillus cereus, and Pseudomonas fluorescens, and the fungi Stachybotrys chartarum, Aspergillus versicolor, and Penicillium spinulosum. Fungi and bacteria generally benefited from the presence of the amoebae, whereas the growth of amoebae was hindered by Streptomyces californicus, Stachybotrys chartarum, and Bacillus cereus. Pseudomonas fluorescens slightly enhanced amoebae viability. Amoebae were indifferent to the presence of Aspergillus versicolor and Penicillium spinulosum. Thus, our results show that amoebae can alter the survival and growth of some microbes in moisture-damaged buildings.Key words: moisture-damaged buildings, free-living amoebae, bacteria, fungi, co-culture.
Fhua and HgbA, outer membrane proteins ofActinobacilluspleuropneumoniae: their role as virulence determinants
Authors: Shakarji Lara , Mikael Leonie G , Srikumar Ramakrishnan , Kobisch Marylène , Coulton James W , Jacques Mario
Published: May 2006

For the recently described serotype 15 of biotype I and serotypes 13 and 14 of biotype II of Actinobacillus pleuropneumoniae, fhuA and hgbA were detected by polymerase chain reaction and DNA sequencing. To determine the substrate specificity of the iron receptors FhuA and HgbA and to study their role in the virulence of A. pleuropneumoniae, we used two isogenic A. pleuropneumoniae serotype 1 deletion mutants of fhuA and hgbA. Different sources of iron and siderophores were tested in growth promotion assays. FhuA and HgbA are specific for their ligands ferrichrome and hemoglobin, respectively. The virulence of the two deletion mutant strains was evaluated in experimental infections using specific pathogen-free piglets. While the fhuA mutant (DG02) was as highly virulent as the parental strain S4074, the virulence of the hgbA mutant (ΔhgbA) was reduced. Our data indicate that both FhuA and HgbA are conserved among all serotypes and biotypes of A. pleuropneumoniae and that HgbA, the receptor for porcine hemoglobin, may play a role in virulence.Key words: Actinobacillus pleuropneumoniae, iron uptake, outer membrane receptors, virulence.
Fungicidal properties of two saponins from Capsicum frutescens and the relationship of structure and fungicidal activity
Authors: De Lucca A J , Boue S , Palmgren M S , Maskos K , Cleveland T E
Published: May 2006

Two steroidal saponins have been purified from cayenne pepper (Capsicum frutescens). Both have the same steroidal moiety but differ in the number of glucose moieties: the first saponin has four glucose moieties (molecular mass 1081 Da) and the second contains three glucose moieties (molecular mass 919 Da). Solubility in aqueous solution is less for the saponin containing three glucose moieties than for the one containing four glucose moieties. The larger saponin was slightly fungicidal against the nongerminated and germinating conidia of Aspergillus flavus, A. niger, A. parasiticus, A. fumigatus, Fusarium oxysporum, F. moniliforme, and F. graminearum, whereas, the second saponin (molecular mass 919 Da) was inactive against these fungi. Results indicate that the absence of one glucose molecule affects the fungicidal and aqueous solubility properties of these similar molecules.Key words: fungicidal, saponin, structure–function.
Response surface methodology for the optimization of keratinase production in culture medium containing feathers produced by Kocuria rosea
Authors: Bernal C , Diaz I , Coello N
Published: May 2006

A 43-fold increase in keratinase production by Kocuria rosea was achieved in batch fermentation using response surface methodology. Factorial designs were used to select the components of a culture medium that showed a significant effect on keratinase production. An orthogonal–central composite experimental design was performed, with only two (feathers and magnesium) from nine initial compounds being further analyzed by response surface methodology. An optimum keratinase production of 14 886.9 U/mg was obtained with the following medium composition (per litre): NH4Cl, 0.3 g; NaCl, 0.3 g; K2HPO4, 3.2 g; KH2PO4, 4.0 g; MgSO4·6H2O, 0.5 g; yeast extract, 0.1 g; and finely milled feathers, 30 g. The medium was shaken at 400 r/min with an incubation period of 14 h at 40 °C.Key words: feathers, keratinases, Kocuria rosea, medium optimization, response surface methodology.
Fungal endophyte assemblages from ethnopharmaceutically important medicinal trees
Authors: Tejesvi Mysore V , Mahesh Basavanna , Nalini Monnanda S , Prakash Harishchandra S , Kini Kukkundoor R , Subbiah Ven , Shetty Hunthrike S
Published: May 2006

Endophytic fungi represent an interesting group of microorganisms associated with the healthy tissues of terrestrial plants. They represent a large reservoir of genetic diversity. Fungal endophytes were isolated from the inner bark segments of ethnopharmaceutically important medicinal tree species, namely Terminalia arjuna, Crataeva magna, Azadirachta indica, Holarrhena antidysenterica, Terminalia chebula, and Butea monosperma (11 individual trees), growing in different regions of southern India. Forty-eight fungal species were recovered from 2200 bark segments. Mitosporic fungi represented a major group (61%), with ascomycetes (21%) and sterile mycelia (18%) the next major groups. Species of Fusarium, Pestalotiopsis, Myrothecium, Trichoderma, Verticillium, and Chaetomium were frequently isolated. Exclusive fungal taxa were recovered from five of the six plant species considered for the study of endophytic fungi. Rarefaction indices for species richness indicated the highest expected number of species for bark segments were isolated from T. arjuna and A. indica (20 species each) and from C. magna (18 species).Key words: bark, endophytes, medicinal trees, southern India.
Production of quorum-sensing-related signal molecules by epiphytic bacteria inhabiting wheat heads
Authors: Yoshida Shigenobu , Kinkel Linda L , Shinohara Hirosuke , Numajiri Nobutaka , Hiradate Syuntaro , Koitabashi Motoo , Suyama Kazuo , Negishi Hiromitsu , Tsushima Seiya
Published: May 2006

The production of quorum-sensing-related signal molecules (QSRMs) among culturable bacteria comprising the community on wheat heads was investigated. The taxonomic position of 186 bacterial isolates obtained from ten heads was inferred based on 16S rRNA gene sequences, and their QSRM production was determined using two bioreporter strains of N-acylhomoserine lactones. Approximately 33% of isolates produced QSRMs, though the proportion of QSRM-producing isolates on a wheat head was significantly negatively correlated with population size. Most of the producing isolates were Pantoea species, most commonly Pantoea ananatis. Furthermore, the proportion of Pantoea ananatis that produced QSRMs was significantly negatively correlated with the number of bacterial genera (community richness) on each head. Finally, community richness was positively correlated with population size. Qualitative analysis using thin-layer-chromatography revealed that the QSRMs of Pantoea isolates were composed of at least two compounds. This is the first report indicating that Pantoea ananatis isolates inhabiting wheat heads are capable of producing QSRMs. QSRM production by Pantoea spp. may contribute to the predominance of this genus on wheat heads, particularly at relatively low population densities and community diversity.Key words: quorum sensing, signal molecule, epiphyte, wheat head, Pantoea spp.
Detection of antibiotic-related genes from bacterial biocontrol agents with polymerase chain reaction
Authors: Zhang Y , Fernando W G.D , Kievit T R. de , Berry C , Daayf F , Paulitz T C
Published: May 2006

Pseudomonas chlororaphis PA23, Pseudomonas spp. strain DF41, and Bacillus amyloliquefaciens BS6 consistently inhibit infection of canola petals by Sclerotinia sclerotiorum in both greenhouse and field experiments. Bacillus thuringiensis BS8, Bacillus cereus L, and Bacillus mycoides S have shown significant inhibition against S. sclerotiorum on plate assays. The presence of antibiotic biosynthetic or self-resistance genes in these strains was investigated with polymerase chain reaction and, in one case, Southern blotting. Thirty primers were used to amplify (i) antibiotic biosythetic genes encoding phenazine-1-carboxylic acid, 2,4-diacetylphloroglucinol, pyoluteorin, and pyrrolnitrin, and (ii) the zwittermicin A self-resistance gene. Our findings revealed that the fungal antagonist P. chlororaphis PA23 contains biosynthetic genes for phenazine-1-carboxylic acid and pyrrolnitrin. Moreover, production of these compounds was confirmed by high performance liquid chromatography. Pseudomonas spp. DF41 and B. amyloliquefaciens BS6 do not appear to harbour genes for any of the antibiotics tested. Bacillus thuringiensis BS8, B. cereus L, and B. mycoides S contain the zwittermicin A self-resistance gene. This is the first report of zmaR in B. mycoides.Key words: Pseudomonas, Bacillus, biocontrol, antibiotic genes.
Molecular characterization of microbial communities in Canadian pulp and paper activated sludge and quantification of a novelThiothrix eikelboomii-like bulking filament
Authors: Dumonceaux Tim J , Hill Janet E , Pelletier Carl P , Paice Michael G , Van Kessel Andrew G , Hemmingsen Sean M
Published: May 2006

We examined the microbial community structure and quantified the levels of the filamentous bulking organism Thiothrix eikelboomii in samples of activated sludge mixed liquor suspended solids (MLSS) from Canadian pulp and paper mills. Libraries of chaperonin 60 (cpn60) gene sequences were prepared from MLSS total microbial community DNA and each was compared with cpnDB, a reference database of cpn60 sequences (http://cpndb.cbr.nrc.ca) for assignment of taxonomic identities. Sequences similar to but distinct from the type strain of T. eikelboomii AP3 (ATCC 49788T) (~89% identity over 555 bp) were recovered at high frequency from a mill sample that was experiencing bulking problems at the time of sample collection, which corresponded to microscopic observations using fluorescent in situ hybridization with commercially available 16S rDNA-based probes. We enumerated this strain in five mill-derived MLSS samples using real-time quantitative PCR (qPCR) and found that two samples had high levels of the bulking strain (>1012genomes/g MLSS) and two contained lower but detectable levels of this organism. None of the mill samples contained cpn60 sequences that were identical to the type strain of T. eikelboomii. This technique shows promise for monitoring pulp and paper mill wastewater treatment systems by detecting and enumerating this strain of T. eikelboomii, which may be specific to pulp and paper mill wastewater treatment systems.Key words: activated sludge, biological treatment, bulking; chaperonin 60, cpn60, filamentous bacteria, mixed liquor suspended solids, microbial communities, 021N, qPCR, settling, Thiothrix.
The use of the Mackintosh Probe for site investigation in soft soils
Authors: Fakher A. , Khodaparast M. , Jones C.J.F.P.
Published: May 2006

Dynamic probing can have an important role in geotechnical site investigation. The Mackintosh Probe is a lightweight and portable penetrometer. It is a considerably faster and cheaper tool than boring equipment especially when the depth of exploration is moderate and the soils under investigation are soft or loose. This paper presents the capabilities of the Mackintosh Probe for the investigation of soft deposits. A methodology for the use of the Mackintosh Probe is discussed and the repeatability of test results is studied. Correlations are developed between Mackintosh Probe results and those of the Standard Penetration Test (SPT), as well as, undrained shear strength (
c

u

). The study concludes that the application of the Mackintosh Probe for site investigation in soft deposits is appropriate and cost effective.
Case study of a loess collapse field trial in Kent, SE England
Authors: Zourmpakis A. , Boardman D.I. , Rogers C.D.F. , Jefferson I. , Gunn D.A. , Jackson P.D. , Northmore K.J. , Entwisle D.C. , Nelder L.M. , Dixon N.
Published: May 2006

Loess soils undergo collapse due to bond weakening under loading and, especially, wetting, and consequently constitute a major engineering geology hazard. To understand better the relationship between wetting and volume reduction in loess, a field collapse test was conducted at a ‘brickearth’ quarry, where a 5.0 × 5.0 × 1.5 m deep sample was isolated, flooded in a controlled manner and subjected to a surface stress of up to 210 kPa for 10 days. Geotechnical instrumentation, consisting of piezometers and rod extensometers, was complemented by geophysical instrumentation (resistivity arrays, shear wave transducers and a resistivity probe) to provide evidence of changes in interparticle bonding during the collapse process. Laboratory index and oedometer testing, together with SEM study of samples removed from the site, complemented the site monitoring. The field collapse test eliminated many problems associated with laboratory testing, notably small volumes of material and sample disturbance.

This paper presents the geotechnical findings on ‘large-scale’ loess performance and relates them to the results of shear wave velocity and resistivity monitoring. The different behaviour of two distinct soil strata and the importance of the degree of saturation to soil fabric changes are demonstrated. The results identify how the soil
in situ
and oedometer samples respond under similar applied stresses.
Highly efficient gluten degradation with a newly identified prolyl endoprotease: implications for celiac disease
Authors: Stepniak Dariusz , Spaenij-Dekking Liesbeth , Mitea Cristina , Moester Martine , de Ru Arnoud , Baak-Pablo Renee , van Veelen Peter , Edens Luppo , Koning Frits
Published: May 2006

Celiac disease is a T cell-driven intolerance to wheat gluten. The gluten-derived T cell epitopes are proline-rich and thereby highly resistant to proteolytic degradation within the gastrointestinal tract. Oral supplementation with prolyl oligopeptidases has therefore been proposed as a potential therapeutic approach. The enzymes studied, however, have limitations as they are irreversibly inactivated by pepsin and acidic pH, both present in the stomach. As a consequence, these enzymes will fail to degrade gluten before it reaches the small intestine, the site where gluten induces inflammatory T cell responses that lead to celiac disease. We have now determined the usefulness of a newly identified prolyl endoprotease from Aspergillus niger for this purpose. Gluten and its peptic/tryptic digest were treated with prolyl endoprotease, and the destruction of the T cell epitopes was tested using mass spectrometry, T cell proliferation assays, ELISA, reverse-phase HPLC, SDS-PAGE, and Western blotting. We observed that the A. niger prolyl endoprotease works optimally at 4–5 pH, remains stable at 2 pH, and is completely resistant to digestion with pepsin. Moreover, the A. niger-derived enzyme efficiently degraded all tested T cell stimulatory peptides as well as intact gluten molecules. On average, the endoprotease from A. niger degraded gluten peptides 60 times faster than a prolyl oligopeptidase. Together these results indicate that the enzyme from A. niger efficiently degrades gluten proteins. Future studies are required to determine if the prolyl endoprotease can be used as an oral supplement to reduce gluten intake in patients.
NHE2 is the main apical NHE in mouse colonic crypts but an alternative Na+-dependent acid extrusion mechanism is upregulated in NHE2-null mice
Authors: Guan Yanfang , Dong Jin , Tackett Lixuan , Meyer Jamie W. , Shull Gary E. , Montrose Marshall H.
Published: May 2006

The mechanism of apical Na+-dependent H+ extrusion in colonic crypts is controversial. With the use of confocal microscopy of the living mouse distal colon loaded with BCECF or SNARF-5F (fluorescent pH sensors), measurements of intracellular pH (pHi) in epithelial cells at either the crypt base or colonic surface were reported. After cellular acidification, the addition of luminal Na+ stimulated similar rates of pHi recovery in cells at the base of distal colonic crypts of wild-type or Na+/H+ exchanger isoform 2 (NHE2)-null mice. In wild-type crypts, 20 μM HOE694 (NHE2 inhibitor) blocked 68–75% of the pHi recovery rate, whereas NHE2-null crypts were insensitive to HOE694, the NHE3-specific inhibitor S-1611 (20 μM), or the bicarbonate transport inhibitor 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS; 1 mM). A general NHE inhibitor, 5-( N-ethyl- N-isopropyl)amiloride (EIPA; 20 μM), inhibited pHi recovery in NHE2-null mice (46%) but less strongly than in wild-type mice (74%), suggesting both EIPA-sensitive and -insensitive compensatory mechanisms. Transepithelial Na+ leakage followed by activation of basolateral NHE1 could confound the outcomes; however, the rates of Na+-dependent pHi recovery were independent of transepithelial leakiness to lucifer yellow and were unchanged in NHE1-null mice. NHE2 was immunolocalized on apical membranes of wild-type crypts but not NHE2-null tissue. NHE3 immunoreactivity was near the colonic surface but not at the crypt base in NHE2-null mice. Colonic surface cells from wild-type mice demonstrated S1611- and HOE694-sensitive pHi recovery in response to luminal sodium, confirming a functional role for both NHE3 and NHE2 at this site. We conclude that constitutive absence of NHE2 results in a compensatory increase in a Na+-dependent, EIPA-sensitive acid extruder distinct from NHE1, NHE3, or SITS-sensitive transporters.