Cloning, expression, and nucleotide sequence of the Pseudomonas aeruginosa 142 ohb genes coding for oxygenolytic ortho dehalogenation of halobenzoates

We have cloned and characterized novel oxygenolytic ortho-dehalogenation (ohb) genes from 2-chlorobenzoate (2-CBA)- and 2,4-dichlorobenzoate (2,4-dCBA)-degrading Pseudomonas aeruginosa 142. Among 3,700 Escherichia coli recombinants, two clones, DH5alphaF'(pOD22) and DH5alphaF'(pOD33), converted 2-CBA to catechol and 2,4-dCBA and 2,5-dCBA to 4-chlorocatechol. A subclone of pOD33, plasmid pE43, containing the 3,687-bp minimized ohb DNA region conferred to P. putida PB2440 the ability to grow on 2-CBA as a sole carbon source. Strain PB2440(pE43) also oxidized but did not grow on 2,4-dCBA, 2,5-dCBA, or 2,6-dCBA. Terminal oxidoreductase ISPOHB structural genes ohbA and ohbB, which encode polypeptides with molecular masses of 20,253 Da (beta-ISP) and 48,243 Da (alpha-ISP), respectively, were identified; these proteins are in accord with the 22- and 48-kDa (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) polypeptides synthesized in E. coli and P. aeruginosa parental strain 142. The ortho-halobenzoate 1,2-dioxygenase activity was manifested in the absence of ferredoxin and reductase genes, suggesting that the ISPOHB utilized electron transfer components provided by the heterologous hosts. ISPOHB formed a new phylogenetic cluster that includes aromatic oxygenases featuring atypical structural-functional organization and is distant from the other members of the family of primary aromatic oxygenases. A putative IclR-type regulatory gene (ohbR) was located upstream of the ohbAB genes. An open reading frame (ohbC) of unknown function that overlaps lengthwise with ohbB but is transcribed in the opposite direction was found. The ohbC gene codes for a 48,969-Da polypeptide, in accord with the 49-kDa protein detected in E. coli. The ohb genes are flanked by an IS1396-like sequence containing a putative gene for a 39,715-Da transposase A (tnpA) at positions 4731 to 5747 and a putative gene for a 45,247-Da DNA topoisomerase I/III (top) at positions 346 to 1563. The ohb DNA region is bordered by 14-bp imperfect inverted repeats at positions 56 to 69 and 5984 to 5997.     

Use of both 16S rRNA and engineered functional genes with real-time PCR to quantify an engineered,PCB-degrading Rhodococcus in soil

A real-time PCR (RTm-PCR) assay using fluorescently labeled oligonucleotides (TaqMan probes) was used to detect and quantify the recombinant Rhodococcus sp. strain RHA1(fcb) in soil. One primer and probe set targeted a hypervariable region of the 16S rRNA gene unique to strain RHA1(fcb) and its phylogenetic relatives, and the other set targeted the recombinant 4-chlorobenzoate (4-CBA) degradation operon (fcb) and was strain-specific. The method had a 6-log dynamic range of detection (10(2)-10(7) cells ml(-1)) for both probes when DNA from pure cultures was used. Although the method was less sensitive in soil, the estimated number of cells in soil by real-time PCR corresponded to the measured number of RHA1(fcb) cells determined by colony-forming units.     

Canine sulfotransferase SULT1A1: molecular cloning,expression, and characterization

Sulfotransferases (SULTs) are involved in detoxification and activation of various endogenous and exogenous compounds including important drugs and hormones. SULT1A, the phenol-SULT subfamily, is the most prominent subfamily in xenobiotic metabolism and has been found in several species, e.g., human, rat, and mouse. We have cloned a phenol-sulfating phenol SULT from dog (cSULT1A1) and expressed it in Escherichia coli for characterization. cSULT1A1 showed 85.8, 82.7, 76.3, and 73.6% identities to human P-PST, human M-PST, rat PST-1, and mouse STp1, respectively. It consists of 295 amino acids, which is in agreement with the human ortholog and sulfate substrates typical for the SULT1A family, i.e., p-nitrophenol (PNP), alpha-naphthol, and dopamine. The K(m) for PNP was found to be within the nanomolar range. It also sulfates minoxidil and beta-estradiol but not dehydroepiandrosterone. Western blot analysis indicated that this newly cloned enzyme was found to be ubiquitously expressed in canine tissues with highest expression in male and female liver.     

Deposition of IgM and complement at the dermoepidermal junction in acute and chronic cutaneous graft-vs-host disease in man.

The presence of cutaneous immunoglobulin and complement was investigated in 88 patients with and without graft-vs-host disease (GVHD) after transplantation of bone marrow from HLA identical siblings for the treatment of acute leukemia or aplastic anemia. For comparison, skin biopsies from the patients obtained before transplantation, from 58 healthy individuals (mostly marrow donors) and from four syngeneic marrow recipients were studied. A direct immunfluorescent staining technique was used. Dermo-epidermal IgM deposits were found in 11% of healthy individuals and patients before grafting but were present in 86% of patients with chronic and 39% of patients with acute GVHD. Patients with allogeneic grafts who never had GVHD or who had recovered from it and patients with syngeneic grafts showed findings not different from those in healthy individuals. Findings similar to those with IgM, although less striking, were made for C3, i.e., patients who had chronic or acute GVHD had a high incidence and intensity of C3 deposits at the dermo-epidermal junction. This observation raises the possibility that humoral immunity is involved in the development of GVHD.     

Canine osteosarcoma: Results of amputation with and without adjuvant immunotherapy

Summary Forty-eight dogs with spontaneously occurring osteogenic sarcoma (OS) were treated by amputation. Six dogs died soon after surgery, 34 dogs died or were euthanized 1–11 (median 4) months after, and eight dogs (17%) are alive 11–47 (median 21) months after amputation. Thirty-one dogs did not receive any therapy after amputation. Seventeen dogs received postamputation adjuvant immunotherapy consisting of intradermal bacillus Calmette-Guerin and an autologous tumor homogenate preparation administered every other week for 12 weeks. Twelve of these dogs were included in a prospectively randomized therapeutic trial in which a control group of 11 dogs received no therapy after amputation. Neither disease-free interval nor survival was prolonged by the immunotherapy protocol. Nevertheless, the current study demonstrates the feasibility of studying naturally occurring canine OS as a clinical and therapeutic model for OS in man.     

First report of an arabinose-specific fungal lectin

To date, arabinose-binding lectins have been reported only from the human opportunistic pathogen Pseudomonas aeruginosa, the plant aggressive pathogen Ralstonia solanacearum, and the sponge Pellina semitubulosa. An arabinose-binding lectin with mitogenic activity toward splenocytes and a high specific hemagglutinating activity was isolated in the present study from a wild discomycete mushroom, Peziza sylvestris. The maximal mitogenic activity was induced by a lectin concentration of 8 microM. The lectin was a single-chained protein with a molecular mass of 20 kDa. Its N-terminal sequence showed only slight resemblance to other mushroom lectins. It was adsorbed on both diethylaminoethyl-cellulose and carboxymethyl-cellulose. Unlike previously reported mushroom lectins, the hemagglutinating activity of the lectin was inhibited by arabinose, but not by a large variety of other carbohydrates. The lectin activity was adversely affected in the presence of 0.05 M NaOH or 0.025 M HCl, and when the ambient temperature was elevated above 35 degrees C.     

Expressed sequence tags analysis of Atlantic halibut (Hippoglossus hippoglossus) liver, kidney and spleen tissues following vaccination against Vibrio anguillarum and Aeromonas salmonicida

To investigate the response of Atlantic halibut to vaccination and pathogen exposure, a cDNA library was constructed from liver, kidney and spleen mRNA collected following vaccination against Vibrio anguillarum and Aeromonas salmonicida. After sequencing 1114 clones 1072 (96.23%) readable sequences were obtained of which 106 sequences are the first reported from the fish. Of these, 182 clones (16.98%) contained cell/organism defence genes including immunoglobulin light chain, MHC class I and II, interferon consensus sequence binding protein, B-cell receptor-associated protein, early B-cell factor, 10 complement components, heat shock protein 70 and 90, antimicrobial peptides hepcidin type 1 and 2, and CC chemokine (macrophage inflammatory protein-1 beta-like chemokine, MIP-1beta). Expression of MIP-1beta-like was elevated in the kidney and spleen at 1, 2, 7 and 14 days post vaccination. Functional genes involved in cellular processes of hematopoietic tissues were also identified. These results indicate that this cDNA library contains many important genes involved in the immune response, making it an important resource for studying the response of Atlantic halibut to vaccination or pathogen exposure.     

In silico analysis of ORF1ab in coronavirus HKU1 genome reveals a unique putative cleavage site of coronavirus HKU1 3C-like protease

Recently we have described the discovery and complete genome sequence of a novel coronavirus associated with pneumonia, coronavirus HKU1 (CoV-HKU1). In this study, a detailed in silico analysis of the ORF1ab, encoding the 7,182-amino acid replicase polyprotein in the CoV-HKU1 genome showed that the replicase polyprotein of CoV-HKU1 is cleaved by its papain-like proteases and 3C-like protease (3CL(pro)) into 16 polypeptides homologous to the corresponding polypeptides in other coronaviruses. Surprisingly, analysis of the putative cleavage sites of the 3CL(pro) revealed a unique putative cleavage site. In all known coronaviruses, the P1 positions at the cleavage sites of the 3CL(pro) are occupied by glutamine. This is also observed in CoV-HKU1, except for one site at the junction between nsp10 (helicase) and nsp11 (member of exonuclease family), where the P1 position is occupied by histidine. This amino acid substitution is due to a single nucleotide mutation in the CoV-HKU1 genome, CAG/A to CAT. This probably represents a novel cleavage site because the same mutation was consistently observed in CoV-HKU1 sequences from multiple specimens of different patients; the P2 and P1'-P12' positions of this cleavage site are consistent between CoV-HKU1 and other coronaviruses; and as the helicase is one of the most conserved proteins in coronaviruses, cleavage between nsp10 and nsp11 should be an essential step for the generation of the mature functional helicase. Experiments, including purification and C-terminal amino acid sequencing of the CoV-HKU1 helicase and trans-cleavage assays of the CoV-HKU1 3CL(pro) will confirm the presence of this novel cleavage site.