Impact of 2,4-diacetylphloroglucinol-producing biocontrol strain Pseudomonas fluorescens F113 on intraspecific diversity of resident culturable fluorescent pseudomonads associated with the roots of field-grown sugar beet seedlings

The impact of the 2,4-diacetylphloroglucinol-producing biocontrol agent Pseudomonas fluorescens F113Rif on the diversity of the resident community of culturable fluorescent pseudomonads associated with the roots of field-grown sugar beet seedlings was evaluated. At 19 days after sowing, the seed inoculant F113Rif had replaced some of the resident culturable fluorescent pseudomonads at the rhizoplane but had no effect on the number of these bacteria in the rhizosphere. A total of 498 isolates of resident fluorescent pseudomonads were obtained and characterized by molecular means at the level of broad phylogenetic groups (by amplified ribosomal DNA restriction analysis) and at the strain level (with random amplified polymorphic DNA markers) as well as phenotypically (55 physiological tests). The introduced pseudomonad induced a major shift in the composition of the resident culturable fluorescent Pseudomonas community, as the percentage of rhizoplane isolates capable of growing on three carbon substrates (erythritol, adonitol, and L-tryptophan) not assimilated by the inoculant was increased from less than 10% to more than 40%. However, the pseudomonads selected did not display enhanced resistance to 2,4-diacetylphloroglucinol. The shift in the resident populations, which was spatially limited to the surface of the root (i.e., the rhizoplane), took place without affecting the relative proportions of phylogenetic groups or the high level of strain diversity of the resident culturable fluorescent Pseudomonas community. These results suggest that the root-associated Pseudomonas community of sugar beet seedlings is resilient to the perturbation that may be caused by a taxonomically related inoculant.     

Polymorphism of the polyketide synthase gene phID in biocontrol fluorescent pseudomonads producing 2,4-diacetylphloroglucinol and comparison of PhID with plant polyketide synthases

Many biocontrol fluorescent pseudomonads can protect plants from soilborne fungal pathogens through production of the antifungal secondary metabolite 2,4-diacetylphloroglucinol (Phl). One of the phl biosynthetic genes, phlD, encodes a polyketide synthase similar to plant chalcone synthases. Here, restriction analysis of phlD from 39 Phl+ biocontrol fluorescent pseudomonads yielded seven different banding patterns. The gene was sequenced in seven strains, representing the different restriction patterns. Cluster analysis of phlD restriction data or phlD sequences indicated that phlD polymorphism was high, and two main clusters were obtained when predicted PhlD sequences were compared. When the seven PhlD sequences were studied with those of other procaryotic polyketide synthases (gram-positive bacteria) and plant chalcone synthases, however, Phl+ pseudomonads, gram-positive bacteria, and plants clustered separately. Yet, sequence analysis of active site regions for PhlD and plant chalcone synthases revealed that PhlD can be considered a member of the chalcone synthase family, which may be interpreted as convergent evolution of key enzymes involved in secondary metabolism. For the 39 Phl+ pseudomonads, a relationship was found among phlD restriction patterns, phylogenetic groups defined by 16S rDNA restriction analysis (confirmed by 16S rDNA sequencing), and production levels of Phl in vitro.     

Development of a rapid and convenient method to purify mucins and determine their in vivo synthesis rate in rats

The intestinal mucoprotein synthesis rate was measured in vivo for the first time. For this, a rapid, reproducible, and convenient method to purify mucoproteins from large numbers of intestinal samples at the same time was developed. The method takes advantage of both the high mucin resistance to protease activities due to their extensive glycosylations and the high mucin molecular size. Intestinal homogenates were partially digested with Flavourzyme. Nonprotected proteins partially degraded were easily separated from mucoproteins by small gel filtration chromatography using Sepharose CL-4B. Electrophoretically pure mucins were obtained. Their amino acid composition was typical of purified intestinal epithelial mucins. The mucoprotein synthesis rate was determined in vivo in rats using the flooding dose method with the stable isotope L-[1-13C]valine. Free L-[1-13C]valine enrichments in the intracellular pool were determined by GC-MS. L-[1-13C]valine enrichments into purified mucoproteins or intestinal mucosal proteins were measured by gas chromatography-combustion-isotope ratio mass spectrometry. In rats, we found that the gut mucosa protein synthesis rate (%/day) decreased regularly from duodenum (122%/day) to colon (43%/day). In contrast, mucoprotein fractional synthesis rates were in the same range along the digestive tract, between 112%/day (colon) and 138%/day (ileum).     

Using tolerance induced via the anterior chamber of the eye to inhibit Th2-dependent pulmonary pathology

Anterior chamber-associated immune deviation (ACAID), a manifestation of ocular immune privilege, prevents Th1-dependent delayed hypersensitivity from developing in response to eye-derived Ags, thereby preserving vision. Since Th2-type cells have recently been shown to mediate destructive inflammation of the cornea, we wondered whether pre-emptive induction of ACAID could inhibit Th2 responses. Using a murine model of OVA -specific, Th2-dependent pulmonary inflammation, we pretreated susceptible mice by injecting OVA alone into the anterior chamber, or by injecting OVA-pulsed, TGF-beta2-treated peritoneal exudate cells i.v. These mice were then immunized with OVA plus alum strategy that generates Th2-mediated OVA-specific pulmonary pathology. When pretreated mice were challenged intratracheally with OVA, their bronchoalveolar lavage fluids contained far fewer eosinophils and significantly less IL-4, IL-5, and IL-13 compared with that of positive, nonpretreated controls. Similarly, lung-draining lymph node cells of pretreated mice secreted significantly less IL-4, IL-5, and IL-13 when challenged in vitro with OVA. Moreover, sera from pretreated mice contained much lower titers of OVA-specific IgE Abs. We conclude that Ags injected into the anterior chamber of the eye impair both Th1 and Th2 responses. These results reduce the likelihood that ACAID regulates Th1 responses via a Th2-like mechanism. Thus, immune privilege of the eye regulates inflammation secondary to both Th1- and Th2-type immune responses.     

Characterization of a highly pathogenic H5N1 avian influenza A virus isolated from duck meat

Since the 1997 H5N1 influenza virus outbreak in humans and poultry in Hong Kong, the emergence of closely related viruses in poultry has raised concerns that additional zoonotic transmissions of influenza viruses from poultry to humans may occur. In May 2001, an avian H5N1 influenza A virus was isolated from duck meat that had been imported to South Korea from China. Phylogenetic analysis of the hemagglutinin (HA) gene of A/Duck/Anyang/AVL-1/01 showed that the virus clustered with the H5 Goose/Guandong/1/96 lineage and 1997 Hong Kong human isolates and possessed an HA cleavage site sequence identical to these isolates. Following intravenous or intranasal inoculation, this virus was highly pathogenic and replicated to high titers in chickens. The pathogenesis of DK/Anyang/AVL-1/01 virus in Pekin ducks was further characterized and compared with a recent H5N1 isolate, A/Chicken/Hong Kong/317.5/01, and an H5N1 1997 chicken isolate, A/Chicken/Hong Kong/220/97. Although no clinical signs of disease were observed in H5N1 virus-inoculated ducks, infectious virus could be detected in lung tissue, cloacal, and oropharyngeal swabs. The DK/Anyang/AVL-1/01 virus was unique among the H5N1 isolates in that infectious virus and viral antigen could also be detected in muscle and brain tissue of ducks. The pathogenesis of DK/Anyang/AVL-1/01 virus was characterized in BALB/c mice and compared with the other H5N1 isolates. All viruses replicated in mice, but in contrast to the highly lethal CK/HK/220/97 virus, DK/Anyang/AVL-1/01 and CK/HK/317.5/01 viruses remained localized to the respiratory tract. DK/Anyang/AVL-1/01 virus caused weight loss and resulted in 22 to 33% mortality, whereas CK/HK/317.5/01-infected mice exhibited no morbidity or mortality. The isolation of a highly pathogenic H5N1 influenza virus from poultry indicates that such viruses are still circulating in China and may present a risk for transmission of the virus to humans.     

An integrated, stacked microlaboratory for biological agent detection with DNA and immunoassays

An integrated, stacked microlaboratory for performing automated electric-field-driven immunoassays and DNA hybridization assays was developed. The stacked microlaboratory was fabricated by orderly laminating several different functional layers (all 76 x 76 mm(2)) including a patterned polyimide layer with a flip-chip bonded CMOS chip, a pressure sensitive acrylic adhesive (PSA) layer with a fluidic cutout, an optically transparent polymethyl methacrylate (PMMA) film, a PSA layer with a via, a patterned polyimide layer with a flip-chip bonded silicon chip, a PSA layer with a fluidic cutout, and a glass cover plate layer. Versatility of the stacked microlaboratory was demonstrated by various automated assays. Escherichia coli bacteria and Alexa-labeled protein toxin staphylococcal enterotoxin B (SEB) were detected by electric-field-driven immunoassays on a single chip with a specific-to-nonspecific signal ratios of 4.2:1 and 3.0:1, respectively. Furthermore, by integrating the microlaboratory with a module for strand displacement amplification (SDA), the identification of the Shiga-like toxin gene (SLT1) from E. coli was accomplished within 2.5 h starting from a dielectrophoretic concentration of intact E. coli bacteria and finishing with an electric-field-driven DNA hybridization assay, detected by fluorescently labeled DNA reporter probes. The integrated microlaboratory can be potentially used in a wide range of applications including detection of bacteria and biowarfare agents, and genetic identification.     

Dopamine D1 receptors in the gills of Chinese crab Eriocheir sinensis

Our investigations indicate the existence of binding sites for [3H]SCH23390 on crude membrane preparations in the anterior and posterior gills of the Chinese crab, Eriocheir sinensis acclimated to freshwater (FW) or seawater (SW). Maximum specific binding in the posterior gills is always higher than in the anterior gills, independent of saline acclimation. Kd values are similar in the two regions, suggesting the same affinity of both types of gills for the ligand, either from FW or from SW crabs.     

Cloning and expression of a heme binding protein from the genome of Saccharomyces cerevisiae

The YLR205c gene of Saccharomyces cerevisiae does not show significant sequence identity to any known gene, except for heme oxygenase (22% to human HO-1). The YLR205 ORF was cloned and overexpressed in both Escherichia coli and S. cerevisiae. Both expression systems yielded proteins that bound heme tightly. The isolated YLR205c protein underwent reduction in the presence of either NADPH-cytochrome P450 reductase or NADH-putidaredoxin-putidaredoxin reductase but did not exhibit heme oxygenase activity. The protein exhibited modest H(2)O(2)-dependent peroxidase activities with guaiacol, potassium iodide, and 2,2(')-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS). Thus, YLR205c codes for a hemoprotein of unknown physiological function that exhibits peroxidase activity.     

A C-terminal region of RAG1 contacts the coding DNA during V(D)J recombination

The site-specific DNA rearrangement process, called V(D)J recombination, creates much of the diversity of immune receptor molecules in the adaptive immune system. Central to this reaction is the organization of the protein-DNA complex containing the proteins RAG1 and RAG2 and their DNA targets. A long-term goal is to appreciate the three-dimensional relationships between the proteins and DNA that allow the assembly of the appropriate reaction intermediates, resulting in concerted cleavage and directed rejoining of the DNA ends. Previous cross-linking approaches have mapped RAG1 contacts on the DNA. RAG1 protein contacts the DNA at the conserved heptamer and nonamer sequences as well as at the coding DNA adjacent to the heptamer. Here we subject RAG1, covalently cross-linked to DNA substrates, to partial cyanogen bromide degradation or trypsin proteolysis in order to map contacts on the protein. We find that coding-sequence contacts occur near the C terminus of RAG1, while contacts made within the recombination signal sequence occur nearer the N terminus of the core region of RAG1. A deletion protein lacking the C-terminal DNA-contacting region is still capable of making the N-terminal contacts. This suggests that the two binding interactions may exist on two separate domains of the protein. A trypsin cleavage pattern of the native protein supports this conclusion. A two-domain model for RAG1 is evaluated with respect to the larger recombination complex.     

Identification of a fourth subunit of mammalian DNA polymerase delta

A 12-kDa and two 25-kDa polypeptides were isolated with highly purified calf thymus DNA polymerase delta by conventional chromatography. A 16-mer peptide sequence was obtained from the 12-kDa polypeptide which matched a new open reading frame from a human EST () encoding a hypothetical protein of unknown function. The protein was designated as p12. Human EST was identified as the putative human homologue of Schizosaccharomyces pombe Cdm1 by a tBlastn search of the EST data base using S. pombe Cdm1. The open reading frame of human EST encoded a polypeptide of 107 amino acids with a predicted molecular mass of 12.4 kDa, consistent with the experimental findings. p12 is 25% identical to S pombe Cdm1. Both of the 25-kDa polypeptide sequences matched the hypothetical KIAA0039 protein sequence, recently identified as the third subunit of pol delta. Western blotting of immunoaffinity purified calf thymus pol delta revealed the presence of p125, p50, p68 (the KIAA0039 product), and p12. With the identification of p12 mammalian pol delta can now be shown to consist of four subunits. These studies pave the way for more detailed analysis of the possible functions of the mammalian subunits of pol delta.