Identification of atypical Aeromonas salmonicida: inter-laboratory evaluation and harmonization of methods

Estrogen therapy increases plasma HDL levels, which may reduce cardiovascular risk in postmenopausal women. The mechanism of action of estrogen in influencing various steps in hepatic HDL and apolipoprotein (apo) A-I synthesis and secretion are not fully understood. In this study, we have used the human hepatoblastoma cell line (Hep G2) as an in vitro model system to delineate the effect of estradiol on multiple regulatory steps involved in hepatic HDL metabolism. Incubation of Hep G2 cells with estradiol resulted in the following statistically significant findings: (1) increased accumulation of apoA-I in the medium without affecting uptake/removal of radiolabeled HDL-protein; (2) accelerated incorporation of [3H]leucine into apoA-I; (3) selective increase in [3H]leucine incorporation into lipoprotein (LP) A-I but not LP A-I+A-II HDL particles (HDL particles without and with apoA-II, respectively); (4) increased ability of apoA-I-containing particles to efflux cholesterol from fibroblasts; (5) stimulated steady state apoA-I but not apoA-II mRNA expression; and (6) increased newly transcribed apoA-I mRNA message without effect on apoA-I mRNA half-life. The data indicate that estradiol stimulates newly transcribed hepatic apoA-I mRNA, resulting in a selective increase in LP A-I, a subfraction of HDL that is associated with decreased atherosclerotic cardiovascular disease, especially in premenopausal women.     

Cloning and characterization of two rhamnogalacturonan hydrolase genes from Aspergillus niger

A rhamnogalacturonan hydrolase gene of Aspergillus aculeatus was used as a probe for the cloning of two rhamnogalacturonan hydrolase genes of Aspergillus niger. The corresponding proteins, rhamnogalacturonan hydrolases A and B, are 78 and 72% identical, respectively, with the A. aculeatus enzyme. In A. niger cultures which were shifted from growth on sucrose to growth on apple pectin as a carbon source, the expression of the rhamnogalacturonan hydrolase A gene (rhgA) was transiently induced after 3 h of growth on apple pectin. The rhamnogalacturonan hydrolase B gene was not induced by apple pectin, but the rhgB gene was derepressed after 18 h of growth on either apple pectin or sucrose. Gene fusions of the A. niger rhgA and rhgB coding regions with the strong and inducible Aspergillus awamori exlA promoter were used to obtain high-producing A. awamori transformants which were then used for the purification of the two A. niger rhamnogalacturonan hydrolases. High-performance anion-exchange chromatography of oligomeric degradation products showed that optimal degradation of an isolated highly branched pectin fraction by A. niger rhamnogalacturonan hydrolases A and B occurred at pH 3.6 and 4.1, respectively. The specific activities of rhamnogalacturonan hydrolases A and B were then 0.9 and 0.4 U/mg, respectively, which is significantly lower than the specific activity of A. aculeatus rhamnogalacturonan hydrolase (2.5 U/mg at an optimal pH of 4.5). Compared to the A enzymes, the A. niger B enzyme appears to have a different substrate specificity, since additional oligomers are formed.     

Isolation of Aeromonas salmonicida in association with purple-pigmented bacteria in sediment from a Scottish loch

Aeromonas salmonicida was recovered in close association with an unidentified purple-pigmented organism, which was isolated from sediment in a Scottish loch during November (1997) and February (1998). However, there has not been any evidence of A. salmonicida infections, specifically furunculosis, associated with the fish in this loch.     

Cloning and characterization of two immunophilin-like genes, ilpA and fkpA, on a single 3.9-kilobase fragment of Aeromonas hydrophila genomic DNA

Antiserum to Aeromonas hydrophila A6 cell envelopes was shown in a previous study (C. Y. F. Wong, G. Mayrhofer, M. W. Heuzenroeder, H. M. Atkinson, D. M. Quinn, and R. L. P. Flower, FEMS Immunol. Med. Microbiol. 15:233-241, 1996) to protect mice against lethal infection by this organism. In this study, colony blot analysis of an A. hydrophila genomic library using antiserum to A. hydrophila A6 cell envelopes revealed a cosmid clone expressing a 30-kDa protein which has not been described previously in aeromonads. The nucleotide sequence of a 3.9-kb fragment derived from this cosmid which expressed the 30-kDa protein revealed two potential open reading frames (ORFs) with homology to known immunophilin proteins. ORF1 encoded a 212-amino-acid protein (molecular mass, 22.4 kDa) with 56% identity to the immunophilin SlyD protein of Escherichia coli. ORF1 was subsequently designated ilpA (immunophilin-like protein). ORF3 encoded a potential gene product of 268 amino acids with a typical signal sequence and a predicted molecular size of 28.7 kDa. The inferred amino acid sequence showed 46% identity with the sequence of the FkpA protein of E. coli and 40% identity with the sequence of the macrophage infectivity potentiator (Mip) protein of Legionella pneumophila. ORF3 was designated fkpA (FK506 binding protein) by analogy with the E. coli FkpA protein. Expression of the FkpA protein was confirmed by Western blot (immunoblot) analysis, which detected a 30-kDa protein, with antiserum to the Mip protein of Legionella longbeachae and a specific antiserum to anA. hydrophila 30-kDa membrane protein. PCR and Southern analysis showed that a DNA sequence encoding FkpA was found in all 178 aeromonads of diverse origins tested. A nonpolar insertion mutation in the fkpA gene did not attenuate virulence in a suckling mouse model nor did it affect the expression of hemolysins or DNase. This suggests that either the fkpA gene is not essential in the virulence of A. hydrophila under these conditions or there are other genes in A. hydrophila coding for proteins with similar functions.     

Cloning and characterization of two genes encoding dihydroxyacetone kinase from Schizosaccharomyces pombe IFO 0354

We report the cloning and characterization of two genes encoding dihydroxyacetone kinase (EC 2.7.1.29), SpDAK1 and SpDAK2, from Schizosaccharomyces pombe IFO 0354. The open reading frames of both genes encode 591 amino acids and have Mrs of 62158 and 62170, respectively. Both predicted amino acid sequences exhibited a high identity to each other (99.8%) and relatively high identities (30% to 76%) to other putative dihydroxyacetone kinase gene products. A Western blot analysis showed that these enzymes are induced by glycerol and repressed by glucose. A genomic Southern blot analysis indicated the presence of SpDAK1 and the absence of SpDAK2 in a standard laboratory strain, S. pombe 972h-.     

Identification and characterization of two thrombin-activatable fibrinolysis inhibitor isoforms

OBJECT: The indications, operative findings, and outcomes of vestibular schwannoma microsurgery are controversial when it is performed after stereotactic radiosurgery. To address these issues, the authors reviewed the experience at two academic medical centers. METHODS: During a 10-year interval, 452 patients with unilateral vestibular schwannomas underwent gamma knife radiosurgery. Thirteen patients (2.9%) underwent delayed microsurgery at a median of 27 months (range 7-72 months) after they had undergone radiosurgery. Six of the 13 patients had undergone one or more microsurgical procedures before they underwent radiosurgery. The indications for surgery were tumor enlargement with stable symptoms in five patients, tumor enlargement with new or increased symptoms in five patients, and increased symptoms without evidence of tumor growth in three patients. Gross-total resection was achieved in seven patients and near-gross-total resection in four patients. The surgery was described as more difficult than that typically performed for schwannoma in eight patients, no different in four patients, and easier in one patient. At the last follow-up evaluation, three patients had normal or near-normal facial function, three patients had moderate facial dysfunction, and seven had facial palsies. Three patients were incapable of caring for themselves, and one patient died of progression of a malignant triton tumor. CONCLUSIONS: Failed radiosurgery in cases of vestibular schwannoma was rare. No clear relationship was demonstrated between the use of radiosurgery and the subsequent ease or difficulty of delayed microsurgery. Because some patients have temporary enlargement of their tumor after radiosurgery, the need for surgical resection after radiosurgery should be reviewed with the neurosurgeon who performed the radiosurgery and should be delayed until sustained tumor growth is confirmed. A subtotal tumor resection should be considered for patients who require surgical resection of their tumor after vestibular schwannoma radiosurgery.     

Expression and characterization of the recombinant gene encoding chitinase from Aeromonas caviae

The gene encoding a chitinase from Aeromonas caviae was cloned by PCR techniques. Its recombinant gene expression was performed using pET20b(+) in Escherichia coli BL21 (DE3). The recombinant chitinase with the extra 33 and 13 amino acids in its N- and C-termini, respectively, was purified to near homogeneity using His-Tag affinity chromatography. The recombinant chitinase was found to be present in both the culture medium and the cytoplasm. A single protein band on the native polyacrylamide gel was confirmed by both the activity staining and protein staining. The optimum pH and temperature of the recombinant chitinase were determined to be 6.25-6.5 and 42.5 degrees C, respectively. It was stable within the pH range of 5-7. Significant activity stimulation by Cu2+ and inhibition by Fe3+ and Hg2+ were observed. Detergents such as SDS and Triton X-100 strongly inhibited the enzyme activity. Substrates such as 4-methylumbelliferyl-N,N'-diacetylchitobioside and 4-methylumbelliferyl-N,N',N"-triacetylchitotriose were hydrolyzed by the recombinant chitinase; however, 4-methylumbelliferyl-N-acetylglucosaminide was not cleaved during the activity assay periods. When chitin power was suspended in buffer with the chitinase (pH 6.5 and 42.5 degrees C), N-acetylchitooligosaccharides [(GlcNAc)n, n = 1-4] were detected at 24 h.     

Identification and structural characterization of two genes encoding glutamate transporter homologues differently expressed in the nervous system of Drosophila melanogaster

The aim of the present investigation was to study the effect of neurotoxic ibotenic acid lesion of the retrochiasmatic area on the daily profile of pineal N-acetylserotonin and melatonin synthesis and on the pineal metabolic reactivity to nocturnal short-term retinal photostimulation. Groups of rats were killed 6 h after lights off either in the dark of immediately after being photostimulated for 1 or 15 min. Additionally, groups of rats were sacrificed at six different time points throughout the 24-hour light-dark cycle. The results suggested the presence of two functionally distinct territories in the retrochiasmatic area. The basal retrochiasmatic area, an area situated immediately ventral to the third ventricle, behind the suprachiasmatic nuclei and in front of the arcuate nucleus, is implicated in the nocturnal inhibitory process induced by short-term retinal photostimulation. The lateral retrochiasmatic area, which is situated immediately lateral to the anterior periventricular nucleus, below the anterior hypothalamic nucleus and in front of the ventromedial hypothalamic nucleus, is importantly involved in the control of the peak amplitude of the daily production of N-acetylserotonin and melatonin by the pineal gland.     

Identification and molecular characterization of the p24 dynactin light chain

Intracellular transport along microtubules uses the motor proteins cytoplasmic dynein and kinesin. Cytoplasmic dynein is responsible for movement to the minus ends of microtubules and the evidence indicates that dynein interacts with another protein complex, dynactin. In order to better understand how these proteins function, we have sought to identify and clone the subunit polypeptides of these two complexes, in particular their light chains. Dynactin is made up of eight subunits of approximately 24,000 to 160,000 Da. In order to clone the p24 subunit, the components of purified dynactin were resolved by SDS polyacrylamide gel electrophoresis. The amino acid sequence of a tryptic peptide from the 24,000-Mr region of the gel was obtained and a candidate polypeptide identified by a screen of the databases. This polypeptide has a predicted molecular weight of 20,822 Da. Using an antibody to a different region of this protein, we demonstrate that it copurifies with microtubules and elutes from the microtubule pellet with characteristics similar to those of the dynactin complex and distinct from those of cytoplasmic dynein. This polypeptide co-sediments with dynactin on sucrose density gradients and it also co-immunoprecipitates with dynactin, but not with kinesin or cytoplasmic dynein. Together these results demonstrate that this polypeptide is the p24 subunit of dynactin. Analysis of the predicted amino acid sequence of p24 shows that it is a unique protein that has no significant similarity to known enzymes or other proteins. Structural analysis indicates that most of this protein will form an alpha-helix and that portions of the molecule may participate in the formation of coiled-coils. Since stoichiometric analysis of dynactin indicates that there is one molecule of p24 per dynactin complex, these characteristics suggest that this polypeptide may be involved in protein-protein interactions, perhaps in the assembly of the dynactin complex.     

Identification and characterization of the glycoprotein E and I genes of canine herpesvirus

We have determined the sequence of the gE and gI genes of canine herpesvirus (CHV), DFD-6 strain. The gE ORF codes for a 522 a.a. polypeptide with a signal sequence at the amino-terminus and a trans-membrane domain at the carboxy-terminus. The gI ORF codes for a 259 a.a. polypeptide with a signal sequence but no trans-membrane domain. Comparison with another line of CHV indicated that the DFD-6 gI gene underwent a frame-shift mutation which caused the loss of the trans-membrane domain. Antibodies against the gE and gI polypeptides detected a 94 kDa gE and a broad band of gI (55-62 kDa) in DFD-6 infected cells, respectively. The precursor of DFD-6 gE is modified to the mature form by N-linked glycosylation only in the presence of gI. Together with the fact that the gI- mutant of DFD-6 produced smaller plaques, it is suggested that the truncated DFD-6 gI is functional. The precursor of DFD-6 gI is modified to the mature form by N-linked glycosylation only in the presence of gE.     

Identification of LytSR-regulated genes from Staphylococcus aureus

In this report, the characterization of a Staphylococcus aureus operon containing two LytSR-regulated genes, lrgA and lrgB, is described. Sequence and mutagenesis studies of these genes suggest that lrgA encodes a murein hydrolase exporter similar to bacteriophage holin proteins while lrgB may encode a protein having murein hydrolase activity.     

The role of the capsular polysaccharide of Aeromonas salmonicida in the adherence and invasion of fish cell lines

The ability of several Aeromonas salmonicida strains grown under different conditions (capsulated and non-capsulated) to adhere to and invade two fish cell lines was compared. The level of adherence was slightly higher when the strains were grown under conditions promoting capsule formation than when the same strains were grown under conditions which did not promote capsule formation. However, the most significant difference among the wild-type strains grown under conditions promoting capsule formation was the ability to invade fish cell lines, which was significantly higher than when the same strains were grown under conditions which did not promote capsule formation. From these results we conclude that the capsular polysaccharide, in these strains, is an important factor for intracellular invasion.     

Intronic U14 snoRNAs of Xenopus laevis are located in two different parent genes and can be processed from their introns during early oogenesis

U14 is a member of the rapidly growing family of intronic small nucleolar RNAs (snoRNAs) that are involved in pre-rRNA processing and ribosome biogenesis. These snoRNA species are encoded within introns of eukaryotic protein coding genes and are synthesized via an intron processing pathway. Characterization of Xenopus laevis U14 snoRNA genes has revealed that in addition to the anticipated location of U14 within introns of the amphibian hsc70 gene (introns 4, 5 and 7), additional intronic U14 snoRNAs are also found in the ribosomal protein S13 gene (introns 3 and 4). U14 is thus far a unique intronic snoRNA in that it is encoded within two different parent genes of a single organism. Northern blot analysis revealed that U14 snoRNAs accumulate during early oocyte development and are rapidly expressed after the mid-blastula transition of developing embryos. Microinjection of hsc70 pre-mRNAs into developing oocytes demonstrated that oocytes as early as stages II and III are capable of processing U14 snoRNA from the pre-mRNA precursor. The ability of immature oocytes to process intronic snoRNAs is consistent with the observed accumulation of U14 during oocyte maturation and the developmentally regulated synthesis of rRNA during oogenesis.     

Purification and characterization of enterotoxin produced by Aeromonas sobria

We purified the toxin of Aeromonas sobria capable of inducing a positive response in the mouse intestinal loop assay. The purified toxin showed a positive response not only in the loop assay but also in a hemolytic assay. Subsequently, we cloned the toxin gene and demonstrated that the product of this gene possessed both hemolytic and enterotoxic activities. These results showed that the enterotoxin of A. sobria possesses hemolytic activity. Nucleotide sequence determination of the toxin gene and amino acid sequence analysis of the purified toxin revealed that it is synthesized as a precursor composed of 488 amino acid residues, and that the 24 amino-terminal amino acid residues of the precursor is removed in the mature toxin. As antiserum against the purified toxin neutralized the fluid accumulation induced by living cells not only of A. sobria but also of A. hydrophila, this and antigenically related toxin(s) are thought to play an essential role in the induction of diarrhea by these organisms. The toxin-injured Chinese hamster ovary (CHO) cells induced the release of intracellular lactose dehydrogenase (LDH). The release of LDH from CHO cells and the lysis of erythrocytes by the toxin were repressed by the addition of dextran to the reaction solution, indicating that the toxin forms pores in the membranes and that the cells were injured by the osmotic gradient developed due to pore formation. However, the histopathological examination of intestinal cells exposed to the toxin showed that it caused fluid accumulation in the mouse intestinal loop without causing cellular damage.     

Identification and characterization of genes that interact with lin-12 in Caenorhabditis elegans

We identified and characterized 14 extragenic mutations that suppressed the dominant egg-laying defect of certain lin-12 gain-of-function mutations. These suppressors defined seven genes: sup-17, lag-2, sel-4, sel-5, sel-6, sel-7 and sel-8. Mutations in six of the genes are recessive suppressors, whereas the two mutations that define the seventh gene, lag-2, are semi-dominant suppressors. These suppressor mutations were able to suppress other lin-12 gain-of-function mutations. The suppressor mutations arose at a very low frequency per gene, 10-50 times below the typical loss-of-function mutation frequency. The suppressor mutations in sup-17 and lag-2 were shown to be rare non-null alleles, and we present evidence that null mutations in these two genes cause lethality. Temperature-shift studies for two suppressor genes, sup-17 and lag-2, suggest that both genes act at approximately the same time as lin-12 in specifying a cell fate. Suppressor alleles of six of these genes enhanced a temperature-sensitive loss-of-function allele of glp-1, a gene related to lin-12 in structure and function. Our analysis of these suppressors suggests that the majority of these genes are part of a shared lin-12/glp-1 signal transduction pathway, or act to regulate the expression or stability of lin-12 and glp-1.     

The plasmid profiles of fish pathogenic isolates of Aeromonas salmonicida, Vibrio anguillarum, and Vibrio ordalii from the Atlantic and Pacific coasts of Canada

The plasmid profiles of oxytetracycline- and streptomycin-resistant isolates of Aeromonas salmonicida, Vibrio anguillarum, and Vibrio ordalii were examined by agarose gel electrophoresis. Bacterial isolates were from disease outbreaks in fish on the Atlantic and Pacific coasts. Resistant isolates were examined when grown in the presence and absence of antibiotic. Alkaline lysis methods were used for plasmid isolation. Vibrio spp. were predominantly plasmidless, except for a 47-kilobase (kb) plasmid. Atlantic coast isolates of A. salmonicida possessed four or six plasmids, with four smaller plasmids ranging in size from 4.3 to 8.1 kb being consistently observed. The plasmid profiles of antibiotic-sensitive ATCC strains were identical. The plasmid profiles of the Pacific coast isolates of A. salmonicida varied slightly from those of the Atlantic coast isolates with six plasmids observed, ranging in size from 4.2 to 8.9 kb. Resistance to the antibiotics was not altered following plasmid curing experiments and resistance was not transferable to Escherichia coli. Thus, resistance to oxytetracycline and streptomycin did not appear to be plasmid mediated.