Current production by non-methanotrophic bacteria enriched from an anaerobic methane-oxidizing microbial community.

In recent years, the externalization of electrons as part of respiratory metabolic processes has been discovered in many different bacteria and some archaea. Microbial extracellular electron transfer (EET) plays an important role in many anoxic natural or engineered ecosystems. In this study, an anaerobic methane-converting microbial community was investigated with regard to its potential to perform EET. At this point, it is not well-known if or how EET confers a competitive advantage to certain species in methane-converting communities. EET was investigated in a two-chamber electrochemical system, sparged with methane and with an applied potential of +400 mV versus standard hydrogen electrode. A biofilm developed on the working electrode and stable low-density current was produced, confirming that EET indeed did occur. The appearance and presence of redox centers at -140 to -160 mV and at -230 mV in the biofilm was confirmed by cyclic voltammetry scans. Metagenomic analysis and fluorescence in situ hybridization of the biofilm showed that the anaerobic methanotroph 'Candidatus Methanoperedens BLZ2' was a significant member of the biofilm community, but its relative abundance did not increase compared to the inoculum. On the contrary, the relative abundance of other members of the microbial community significantly increased (up to 720-fold, 7.2% of mapped reads), placing these microorganisms among the dominant species in the bioanode community. This group included Zoogloea sp., Dechloromonas sp., two members of the Bacteroidetes phylum, and the spirochete Leptonema sp. Genes encoding proteins putatively involved in EET were identified in Zoogloea sp., Dechloromonas sp. and one member of the Bacteroidetes phylum. We suggest that instead of methane, alternative carbon sources such as acetate were the substrate for EET. Hence, EET in a methane-driven chemolithoautotrophic microbial community seems a complex process in which interactions within the microbial community are driving extracellular electron transfer to the electrode.

Different divalent cation requirements for binding IgM complexes to lymphocytes and macrophages.

We have found that Mg++ supports IgM complex binding to mouse lymphocytes but not to macrophages. In contrast, Ca++ supports IgM complex binding to macrophages but not lymphocytes. IgG complex binding to both lymphocytes and macrophages is divalent cation independent. These findings allow one to distinguish IgG from IgM binding and macrophage from lymphocyte IgM complex binding by their differential divalent cation requirements.

Recommendations for the treatment of established fungal infections.

Evidence-based guidelines for the treatment of established fungal infections in the adult haematology/oncology setting were developed by a national consensus working group representing clinicians, pharmacists and microbiologists. These updated guidelines replace the previous guidelines published in the Internal Medicine Journal by Slavin et al. in 2004. The guidelines are pathogen-specific and cover the treatment of the most common fungal infections including candidiasis, aspergillosis, cryptococcosis, zygomycosis, fusariosis, scedosporiosis, and dermatophytosis. Recommendations are provided for management of refractory disease or salvage therapies, and special sites of infections such as the cerebral nervous system and the eye. Because of the widespread use newer broad-spectrum triazoles in prophylaxis and empiric therapy, these guidelines should be implemented in concert with the updated prophylaxis and empiric therapy guidelines published by this group.

Role of protein synthesis in decay and accumulation of mRNA during spore germination in the cellular slime mold Dictyostelium discoideum.

Spore germination in Dictyostelium discoideum is a particularly suitable model for studying the regulation of gene expression, since developmentally regulated changes in both protein and mRNA synthesis occur during the transition from dormant spore to amoeba. The previous isolation of three cDNA clones specific for mRNA developmentally regulated during spore germination allowed for the quantitation of the specific mRNAs during this process. The three mRNAs specific to clones pLK109, pLK229, and pRK270 have half-lives much shorter (minutes) than those of constitutive mRNAs (hours). Using spore germination as a model, we studied the roles of ribosome-mRNA interactions and protein synthesis in mRNA degradation by using antibiotics that inhibit specific reactions in protein biosynthesis. Cycloheximide inhibits the elongation step of protein synthesis. Polysomes accumulate in inhibited cells because ribosomes do not terminate normally and new ribosomes enter the polysome, eventually saturating the mRNA. Pactamycin inhibits initiation, and consequently polysomes break down in the presence of this drug. Under this condition, the mRNA is essentially free of ribosomes. pLK109, pLK229, and pRK270 mRNAs were stabilized in the presence of cycloheximide, but pactamycin had no effect on their normal decay. Since it seems likely that stability of mRNA reflects the availability of sites for inactivation by nucleases, it follows that in the presence of cycloheximide, these sites are protected, presumably by occupancy by ribosomes. No ribosomes are bound to mRNA in the presence of pactamycin, and therefore mRNA degrades at about the normal rate. The data further indicate that a labile protein is probably not involved in mRNA decay or stabilization, since protein synthesis is inhibited equally by both antibiotics. We conclude that it may be important to use more than one type of protein synthesis inhibitor to evaluate whether protein synthesis is required for mRNA decay. The effect of protein synthesis inhibition on mRNA synthesis and accumulation was also studied. mRNA synthesis continues in the presence of inhibitors, albeit at a diminished rate relative to that of the uninhibited control.

The Mouse Genome Database (MGD): from genes to mice--a community resource for mouse biology.

The Mouse Genome Database (MGD) forms the core of the Mouse Genome Informatics (MGI) system (http://www.informatics.jax.org), a model organism database resource for the laboratory mouse. MGD provides essential integration of experimental knowledge for the mouse system with information annotated from both literature and online sources. MGD curates and presents consensus and experimental data representations of genotype (sequence) through phenotype information, including highly detailed reports about genes and gene products. Primary foci of integration are through representations of relationships among genes, sequences and phenotypes. MGD collaborates with other bioinformatics groups to curate a definitive set of information about the laboratory mouse and to build and implement the data and semantic standards that are essential for comparative genome analysis. Recent improvements in MGD discussed here include the enhancement of phenotype resources, the re-development of the International Mouse Strain Resource, IMSR, the update of mammalian orthology datasets and the electronic publication of classic books in mouse genetics.

The Mouse Genome Database (MGD): integrating biology with the genome.

The Mouse Genome Database (MGD) is one component of the Mouse Genome Informatics (MGI) system (http://www.informatics.jax.org), a community database resource for the laboratory mouse. MGD strives to provide a comprehensive knowledgebase about the mouse with experiments and data annotated from both literature and online sources. MGD curates and presents consensus and experimental data representations of genetic, genotype (sequence) and phenotype information including highly detailed reports about genes and gene products. Primary foci of integration are through representations of relationships between genes, sequences and phenotypes. MGD collaborates with other bioinformatics groups to curate a definitive set of information about the laboratory mouse and to build and implement the data and semantic standards that are essential for comparative genome analysis. Recent developments in MGD discussed here include an extensive integration of the mouse sequence data and substantial revisions in the presentation, query and visualization of sequence data.

Mouse/human chimeric antibodies to a tumor-associated antigen: biologic activity of the four human IgG subclasses.

Variable region genes from mouse monoclonal antibody 17-1A (gamma 2a kappa) with specificity for human gastrointestinal malignancies have been paired with human immunoglobulin constant region genes (for heavy and light chains) to produce mouse/human chimeric immunoglobulin molecules (chIgG) for each of the four human IgG subclasses. Mouse 17-1A and the four chIgG bound similarly to two human colon cancer cell lines and had comparable binding affinities. The chIgG1 and chIgG3 molecules mediated lymphocyte and monocyte antibody-dependent cell-mediated cytotoxicity (ADCC) to colon cancer tumor cell lines comparable to that of the parent murine 17-1A. The chIgG2 and chIgG4 molecules were able to mediate ADCC to colon cancer cell lines but were clearly inferior to the chIgG1 and chIgG3 reagents. None of the chIgG antibodies or the murine 17-1A was able to mediate complement lysis of colon cancer cell lines. These studies demonstrate the ability to produce all four human IgG subclass chimeric molecules which retain biologic activity. We have confirmed the subclass preferences of human lymphocyte and monocyte Fc receptors for human IgG subclasses previously determined by studies with monomeric or aggregated IgG. These data may aid in the selection of chimeric antibodies for in vivo trials.

Isolation of a Dictyostelium discoideum 14-3-3 homologue.

A 1.0 kb cDNA clone (Dd14-3-3) encoding a 14-3-3 homologue was isolated from a Dictyostelium discoideum cDNA library. The putative Dd14-3-3 protein has highest sequence identity to a barley 14-3-3 isoform (74%). Southern blot analysis suggests that only one 14-3-3 gene is present in the Dictyostelium genome. Highest Dd14-3-3 expression is observed in vegetatively growing cells, and expression decreases during multicellular development. In contrast, Dd14-3-3 protein levels detected immunochemically remained constant during Dictyostelium development. Expression of the Dd14-3-3 cDNA in Saccharomyces cerevisiae complemented the lethal disruption of the two yeast genes encoding 14-3-3 proteins (BMH1 and BMH2). This shows that Dd14-3-3 can fulfil the same function(s) as the yeast 14-3-3 proteins.

Human autoantibodies to carcinoembryonic antigen (CEA) induced by a vaccinia-CEA vaccine.

Carcinoembryonic antigen (CEA) is a well-characterized oncofetal glycoprotein whose overexpression by human adenocarcinomas has been a target for cancer immunotherapy. Limited information is available regarding the ability of patients to mount an antibody response to this self-antigen following vaccination. Recombinant vaccinia viruses encoding full-length or internally deleted cDNAs for human CEA were used to vaccinate 32 patients with CEA-expressing adenocarcinomas, predominantly of colorectal origin. CEA-specific autoantibodies were induced by vaccination in 7 of 32 patients. None of the patients had CEA antibodies detected before vaccination. CEA specificity of the antibodies initially identified by ELISA was confirmed by competitive inhibition analysis as well as recognition of recombinant CEA produced in baculovirus-infected insect cell cultures and human cell cultures by Western blot. The CEA autoantibodies were predominantly IgG1, with a minority of patients also demonstrating IgM autoantibodies. CEA antibodies were of low titer and low avidity, based on competitive inhibition assays. These autoantibodies did not affect clinical serum CEA protein quantitation. Furthermore, elevated serum CEA levels commonly encountered in patients with advanced adenocarcinoma did not hinder detection of low avidity polyclonal CEA antibodies. CEA antibodies such as those induced in these pilot trials are projected to have modest antitumor activity. Thus, additional Phase I/II trials of recombinant vaccinia-CEA with alternative prime-boost approaches and/or augmentation strategies are warranted in an effort to enhance the frequency and avidity of CEA-specific autoantibodies and cytolytic T cells before Phase III trials.

Expression of human endogenous retrovirus k envelope transcripts in human breast cancer.

PURPOSE: We investigated the expression of human endogenous retroviral (HERV) sequences in breast cancer. EXPERIMENTAL DESIGN: Reverse transcription-PCR (RT-PCR) was used to examine expression of the envelope (env) region of ERV3, HERV-E4-1, and HERV-K in breast cancer cell lines, human breast tumor samples, adjacent uninvolved breast tissues, nonmalignant breast tissues, and placenta. Expression of HERV transcripts was confirmed by Northern blot analysis and in situ hybridization (ISH). To evaluate coding potential, amplified HERV sequences were cloned into vectors for expression and sequence analysis. RESULTS: No expression of ERV3 or HERV-E4-1 RNA was detected in the analyzed breast samples. In contrast, HERV-K transcripts were detected in most breast cancer cell lines and many breast tumor tissues. Expression was detected in a small percentage of matched, uninvolved breast tissues and in placentas but not nonmalignant breast tissues. In HERV-K-positive breast cancer tissues, Northern blot analysis demonstrated full-length proviral and spliced env transcripts. ISH demonstrated expression of HERV-K transcripts in breast tumor cells but not in normal or uninvolved breast epithelial cells. Independently isolated clones of HERV-K env cDNA generated recombinant proteins of the expected size. Sequence analysis of env cDNA clones derived from four breast tumor samples revealed >97% identity with the type I HERV-K102, with no premature termination codons. Independent isolates from the same breast tumor sample showed nucleotide sequence differences, suggesting that multiple loci may be transcribed. CONCLUSIONS: These data indicate that HERV-K transcripts with coding potential for the envelope region are expressed frequently in human breast cancer.

Using a tropism-modified adenoviral vector to circumvent inhibitory factors in ascites fluid.

Peritoneal compartmentalization of advanced stage ovarian cancer provides a rational scenario for gene therapy strategies. Several groups are exploring intraperitoneal administration of adenoviral (Ad) vectors for this purpose. We examined in vitro gene transfer in the presence of ascites fluid from ovarian cancer patients and observed significant inhibition of Ad-mediated gene transfer. The inhibitory activity was not identified as either complement or cellular factors, but depletion of IgG from ascites removed the inhibitory activity, implicating neutralizing anti-Ad antibodies. A wide range of preexisting anti-Ad antibody titers in patient ascites fluid was measured by ELISA. Western blot analysis demonstrated that the antibodies were directed primarily against the Ad fiber protein. To circumvent inhibition by neutralizing antibodies, a genetically modified adenoviral vector was tested. The Ad5Luc.RGD vector has an Arg-Gly-Asp (RGD) peptide sequence inserted into the fiber knob domain and enters cells through a nonnative pathway. Compared with the conventional Ad5 vector, Ad5Luc.RGD directed efficient gene transfer to cell lines and primary ovarian cancer cells in the presence of ascites fluid containing high-titer neutralizing anti-Ad antibodies. These results suggest that such modified Ad vectors will be needed to achieve efficient gene transfer in the clinical setting.

SEREX analysis for tumor antigen identification in a mouse model of adenocarcinoma.

Evaluation of immunotherapy strategies in mouse models of carcinoma is hampered by the limited number of known murine tumor antigens (Ags). Although tumor Ags can be identified based on cytotoxic T-cell activation, this approach is not readily accomplished for many tumor types. We applied an alternative strategy based on a humoral immune response, SEREX, to the identification of tumor Ags in the murine colon adenocarcinoma cell line MC38. Immunization of syngeneic C57BL/6 mice with MC38 cells by three different methods induced a protective immune response with concomitant production of anti-MC38 antibodies. Immunoscreening of an MC38-derived expression library resulted in the identification of the endogenous ecotropic leukemia virus envelope (env) protein and the murine ATRX protein as candidate tumor Ags. Northern blot analysis demonstrated high levels of expression of the env transcript in MC38 cells and in several other murine tumor cell lines, whereas expression in normal colonic epithelium was absent. ATRX was found to be variably expressed in tumor cell lines and in normal tissue. Further analysis of the expressed env sequence indicated that it represents a nonmutated tumor Ag. Polynucleotide immunization with DNA encoding the env polypeptide resulted in strong and specific antibody responses to this self Ag in all immunized mice. Thus, SEREX offers a rapid means of identifying tumor Ags in murine cancer models.

Use of UV-irradiated bacteriophage T6 to kill extracellular bacteria in tissue culture infectivity assays.

We have utilized 'lysis from without' mediated by UV-inactivated bacteriophage T6 to eliminate extracellular bacteria in experiments measuring the internalization, intracellular survival and replication of Yersinia pestis within mouse peritoneal macrophages and of Shigella flexneri within a human intestinal epithelial cell line. The technique we describe has the following characteristics: (a) bacterial killing is complete within 15 min at 37 degrees C, with a greater than 10(3)-fold reduction in colony-forming units (CFU); (b) bacteria within cultured mammalian cells are protected from killing by UV-inactivated T6; (c) the mammalian cells are not observably affected by exposure to UV-inactivated T6. This technique has several advantages over the use of antibiotics to eliminate extracellular bacteria and is potentially widely applicable in studies of the interactions between pathogenic bacteria and host phagocytic cells as well as other target tissues.

Hemorrhagic disorder due to an isoniazid-associated acquired factor XIII inhibitor in a patient with Waldenström's macroglobulinemia.

A case is described of a 75-year-old woman with a history of pulmonary tuberculosis and Waldenström's macroglobulinemia who developed an inhibitor of coagulation factor XIII while taking isoniazid. The patient presented with a subcutaneous hematoma of the abdominal wall that extended from the xiphoid process to the symphysis pubis and measured 20 cm in diameter. Results of routine coagulation studies were normal with the exception of an increased solubility of the patient's plasma clot in 5M urea consistent with a deficiency of factor XIII activity. Persistence of the deficiency following a 1:2 dilution of the patient's plasma in normal plasma indicated the presence of an inhibitor. A sample of the patient's plasma was depleted of IgG by streptococcal protein G adsorption. The IgG-depleted plasma did not inhibit factor XIII activity, indicating that the inhibitory activity was not attributable to the underlying IgM paraprotein. The patient's purified IgG, on the other hand, inhibited factor XIII activity and the inhibitory activity could be neutralized by anti-IgG antibody. The patient's IgG also inhibited factor XIII-mediated incorporation of fluorescent monodansylcadaverine into casein. Binding of the patient's IgG to factor XIII concentrate was demonstrated by enzyme-linked immunosorbent assay and the IgG that bound to the factor XIII was demonstrated to be polyclonal. Isoniazid was discontinued after the patient was admitted to the hospital. Cryoprecipitate infusion controlled bleeding and reduced the inhibitor titer by 50%. Treatment with cyclophosphamide and prednisone, followed by extracorporeal immunoadsorption over a staphylococcal protein A column, did not reduce the inhibitor titer further. Plasma exchange therapy reduced the inhibitor titer to undetectable levels but failed to restore factor XIII activity. Infusions of factor XIII concentrate reproducibly restored factor XIII activity and were not associated with an anamnestic rise in the inhibitor titer. This represents the seventh reported case of an acquired inhibitor to factor XIII associated with the ingestion of isoniazid.

3'-Azido-3'-deoxythymidine inhibition of human lymphocyte cytolytic function in vitro.

Despite administration of 3'-azido-3'-deoxythymidine (AZT, Zidovudine) to seriously immunocompromised patients, little has been reported regarding effects of AZT on specific immune functions. This study analyzed the in vitro effect of AZT on normal human lymphocyte cytolytic activity. AZT at concentrations up to 100 microM had no effect when added directly to cytotoxicity assays with lymphocyte effector cells and natural killer (NK)-sensitive or NK-resistant target cells. In contrast, addition of AZT to lymphocytes cultured for 4-10 days with interleukin-2 (IL-2) prior to cytotoxicity assays produced a concentration- and time-dependent inhibition; this effect was not mimicked by acyclovir or ganciclovir. Lymphocyte cell numbers and viability were not reduced in parallel to inhibition of cytolytic activity by AZT. Furthermore, AZT inhibition of IL-2-dependent cytolytic activity was not correlated with alterations in lymphocyte cell surface phenotypes by flow cytometry, and lymphocyte culture supernatant levels of interferon-gamma were not reduced by AZT. These results suggest that AZT may selectively inhibit human lymphocyte functions and thus may have implications for long-term therapeutic administration of AZT in chronic immunodeficiency states.

Effects of synthetic oligonucleotides on human complement and coagulation.

Oligodeoxynucleotide phosphorothioates (PS-oligos) are being studied as novel therapeutic agents based on their ability to inhibit gene expression. Preclinical studies produced unanticipated complement and coagulation effects in monkeys receiving high-dose PS-oligo. In the present in vitro studies, PS-oligo inhibited normal human blood clotting as well as subsequent assays for prothrombin fragment PF(1+2) and hemolytic complement. PS-oligo treatment of normal donor plasma produced concentration-dependent prolongations of clotting times, with the activated partial thromboplastin time more sensitive than prothrombin time or thrombin clotting time. PS-oligo treatment of normal donor serum similarly reduced hemolytic complement activity in a concentration-dependent manner. Reduced hemolysis correlated with increased levels of complement fragment C4d. The anti-heparin drug protamine sulfate inhibited in vitro effects of PS-oligo in both complement and coagulation assays, suggesting that charged residues in internucleotide linkages of PS-oligo mediated the observed activities. Therefore, oligonucleotides with varying internucleotide linkages, nucleotide sequence, or secondary structure were compared. Both complement and coagulation effects appeared to be independent of nucleotide sequence but were strongly related to the nature of internucleotide linkages. Several of these modified oligonucleotides have been shown previously to retain potent antisense activity and thus may represent viable alternatives for antisense therapeutics.

X-ray appearance and clinical significance of left atrial wall calcification.

Ten patients with calcifications of the left atrium are reported with review of the literature. Calcification of the left atrium is frequently associated with history of rheumatic fever, longstanding congestive heart failure, atrial fibrillation, mural thrombus and embolization. Early recognition of such lesions is essential for the management of the patients, particularly when surgical intervention is contemplated. A practical classification of left atrial calcification is proposed according to the dominant lesion in each group: (a) Calcification of the left atrial appendage alone (Mitral stenosis). (b) cacification of all 3 component lesions of the left atrium, i.e., the left atrial appendage, the free wall, and the mitral valve (Severe mitral stenosis). (c) Calcification of the left atrium in MacCallum's patch alone (Mitral insufficiency).

Hepatitis C genotyping by direct sequencing of the product from the Roche AMPLICOR test: methodology and application to a South Australian population.

The Roche AMPLICOR RT-PCR amplifies a 244 nucleotide sequence within the 5' non coding region (5'NCR) of the viral genome and is a widely used commercial test for the qualitative determination of hepatitis C RNA from sera. This paper describes a routine procedure for the purification of the PCR product, and its use in automated DNA sequencing, for determining the genotype of hepatitis C virus (HCV) isolates. Direct sequencing of the purified product was possible for 86% of samples, whilst 14% required additional amplification using a nested PCR method in order to read the resulting electropherogram. This method of genotyping is considerably less expensive than currently available commercial kits, and is convenient for the increasing number of laboratories that have access to automated DNA sequencers. The highly conserved nature of the 5'NCR limited differentiation of types and subtypes to an extent comparable to commercial HCV typing methods. Using this method on available laboratory samples and on patients about to commence interferon therapy, we found a predominance of genotype 1 (59%) and 3a (31%). Analysis of data on the interferon patients showed the median length of time from first exposure to diagnosis to be significantly longer for patients with genotype 1 than genotype 3a.

Cephalosporin utilisation review and evaluation.

The clinical misuse of drugs may result in preventable patient morbidity and mortality, costly remedial care, additional costs for diagnosis and management of iatrogenic disease and unnecessary wastage of healthcare resources. In recognition of this problem, drug utilisation evaluation (DUE) has been recommended as a method for identifying inappropriate or unnecessary drug use and for promoting rational therapy. Growing concern over the widespread misuse of antibiotics, together with the emergence of antimicrobial resistance and escalating expenditures, has resulted in antibiotics being the drugs most frequently chosen for DUE projects. Cephalosporin DUE is well documented as being successful for modifying cephalosporin use and for containing drug expenditure. Studies range from isolated projects to ongoing programmes that comprehensively evaluate cephalosporin use and the impact of corrective strategies. Sensible use of antibiotics requires a clear understanding of the infectious process, the clinical pharmacology of anti-infective agents and an appreciation of clinical and microbiological monitoring and assessment. Audit criteria that incorporate the above principles, and which are described in the studies reviewed in this article, will be useful for other investigators. Through its DUE programme, the Royal Adelaide Hospital has investigated the use of cephalosporins, including ceftriaxone, ceftazidime and cefoxitin. These reviews have resulted in improvements in cephalosporin use and significant cost savings. Alterations to cephalosporin use that were recommended following these reviews have not resulted in adverse changes to post-operative infection rates, clinical outcomes or adverse drug reactions. This experience, combined with that of other investigators, serves as a useful model for the promotion of rational and economical therapy with cephalosporins and other drug groups.