Functional Gene Hybridization Patterns of Terrestrial Ammonia-Oxidizing Bacteria

Abstract The biochemical pathway and genetics of autotrophic ammonia oxidation have been studied almost exclusively in Nitrosomonas europaea. Terrestrial autotrophic ammonia-oxidizing bacteria (AAOs), however, comprise two distinct phylogenetic groups in the beta-Proteobacteria, the Nitrosomonas and Nitrosospira groups. Hybridization patterns were used to assess the potential of functional probes in non-PCR-based molecular analysis of natural AAO populations and their activity. The objective of this study was to obtain an overview of functional gene homologies by hybridizing probes derived from N. europaea gene sequences ranging in size from 0.45 to 4.5 kb, and labeled with 32P to Southern blots containing genomic DNA from four Nitrosospira representatives. Probes were specific for genes encoding ammonia monooxygenase (amoA and amoB), hydroxylamine oxidoreductase (hao), and cytochrome c-554 (hcy). These probes produced hybridization signals, at low stringency (30 degreesC), with DNA from each of the four representatives; signals at higher stringency (42 degreesC) were greatly reduced or absent. The hybridization signals at low stringency ranged from 20 to 76% of the total signal obtained with N. europaea DNA. These results indicate that all four functional genes in the ammonia oxidation pathway have diverged between the Nitrosomonas and Nitrosospira groups. The hao probe produced the most consistent hybridization intensities among the Nitrosospira representatives, suggesting that hao sequences would provide the best probes for non-PCR-based molecular analysis of terrestrial AAOs. Since N. europaea can also denitrify, an additional objective was to hybridize genomic DNA from AAOs with probes for Pseudomonas genes involved in denitrification. These probes were specific for genes encoding heme-type dissimilatory nitrite reductase (dNir), Cu-type dNir, and nitrous oxide reductase (nosz). No hybridization signals were observed from probes for the heme-type dNir or nosz, but Nitrosospira sp. NpAV and Nitrosolobus sp. 24-C hybridized, under low-stringency conditions, with the Cu-type dNir probe. These results indicate that AAOs may also differ in their mechanisms and capacities for denitrification.     

Expression of CA IX in dysplasia adjacent to surgical resection margins of oral squamous cell carcinoma

Carbonic anhydrase (CA) IX is a hypoxia marker located almost exclusively in tumor cells. We analyzed the expression of this marker in dysplastic lesions adjacent to the surgical resection margin in patients with oral squamous cell carcinoma. We investigated 70 archived tumors, 36 of which showed dysplasia adjacent to the surgical margin. We used tissue microarray technology to perform an immunohistochemical study of CA IX expression. We found 12 (33.3%) cases of mild dysplasia (10 negative, 2 positive for CA IX), five (13.9%) cases of moderate dysplasia (3 negative, 2 positive for CA IX), 1 (2.8%) case of severe dysplasia (negative for CA IX) and 18 (50%) cases of carcinoma in situ (10 negative, 8 positive for CA IX). In cases of intense expression of CA IX in the tumor, the same distribution of positive and negative cases was observed in all degrees of dysplasia (mild, moderate, severe), although cases of carcinoma in situ tended to be CA IX positive.     

Feasibility of a liver transcriptomics approach to assess bovine treatment with the prohormone dehydroepiandrosterone (DHEA)

Background  

Within the European Union the use of growth promoting agents in animal production is prohibited. Illegal use of natural prohormones like dehydroepiandrosterone (DHEA) is hard to prove since prohormones are strongly metabolized in vivo. In the present study, we investigated the feasibility of a novel effect-based approach for monitoring abuse of DHEA. Changes in gene expression profiles were studied in livers of bull calves treated orally (PO) or intramuscularly (IM) with 1000 mg DHEA versus two control groups, using bovine 44K DNA microarrays. In contrast to controlled genomics studies, this work involved bovines purchased at the local market on three different occasions with ages ranging from 6 to 14 months, thereby reflecting the real life inter-animal variability due to differences in age, individual physiology, season and diet.     

Autoantibodies specific for apoptotic U1-70K are superior serological markers for mixed connective tissue disease

Modifications occurring on autoantigens during cell death have been proposed to have a role in the initiation of autoimmune diseases. Patients suffering from mixed connective tissue disease (MCTD) produce autoantibodies directed to U1 small nuclear ribonucleoprotein (snRNP), and antibodies against a 70 kDa protein component, the U1-70K (70K) protein, are the most prominent. During apoptosis, 70K is cleaved by caspase-3 to a 40 kDa product, which remains associated with the complex. Autoantibodies preferentially recognizing the apoptotic form of 70K have been described previously, and an apoptosis-specific epitope on 70K has been identified. This study shows that 29 of 53 (54%) MCTD sera preferentially recognize the apoptotic form of 70K over intact 70K. Moreover, we show that antibodies directed to an apoptosis-specific epitope on 70K are more specifically associated with MCTD than other anti-70K antibodies, suggesting that apoptotic 70K is a better antigen for the detection of these antibodies in MCTD patients. Longitudinal analysis of 12 MCTD patients showed in several patients that early sera are relatively enriched with antibodies recognizing an apoptosis-specific epitope, and that the levels of these apoptosis-specific antibodies decrease in time. These findings indicate that the early detection of apoptotic 70K is of considerable interest for anti-U1 snRNP-positive patients.     

Involvement of ethylene and nitric oxide in cell death in mastoparan‐treated unicellular alga Chlamydomonas reinhardtii

This work demonstrates a contribution of ethylene and NO (nitric oxide) in MP (mastoparan)‐induced cell death in the green algae Chlamydomonas reinhardtii. Following MP treatment, C. reinhardtii showed massive cell death, expressing morphological features of PCD (programmed cell death). A pharmacological approach involving combined treatments with MP and ethylene‐ and NO‐interacting compounds indicated the requirement of trace amounts of both ethylene and NO in MP‐induced cell death. By employing a carbon dioxide laser‐based photoacoustic detector to measure ethylene and a QCL (quantum cascade laser)‐based spectrometer for NO detection, simultaneous increases in the production of both ethylene and NO were observed following MP application. Our results show a tight regulation of the levels of both signalling molecules in which ethylene stimulates NO production and NO stimulates ethylene production. This suggests that, in conjunction with the elicitor, NO and ethylene cooperate and act synchronously in the mediation of MP‐induced PCD in C. reinhardtii. To the best of our knowledge, this is the first report on the functional significance of ethylene and NO in MP‐induced cell death.     

Plant‐insect interactions: what can we learn from plant lectins?

Many plant lectins have high anti‐insect potential. Although the effects of most lectins are only moderately influencing development or population growth of the insect, some lectins have strong insecticidal properties. In addition, some studies report a deterrent activity towards feeding and oviposition behavior. Transmission of plant lectins to the next trophic level has been investigated for several tritrophic interactions. Effects of lectins with different sugar specificities can vary substantially with the insect species under investigation and with the experimental setup. Lectin binding in the insect is an essential step in exerting a toxic effect. Attempts have been made to study the interactions of lectins in several insect tissues and to identify lectin‐binding receptors. Ingested lectins generally bind to parts of the insect gut. Furthermore, some lectins such as the Galanthus nivalus agglutinin (GNA) cross the gut epithelium into the hemolymph and other tissues. Recently, several candidate lectin‐binding receptors have been isolated from midgut extracts. To date little is known about the exact mechanism for insecticidal activity of plant lectins. However, insect glycobiology is an emerging research field and the recent technological advances in the analysis of lectin carbohydrate specificities and insect glycobiology will certainly lead to new insights in the interactions between plant lectins and insects, and to a better understanding of the molecular mechanisms involved. © 2010 Wiley Periodicals, Inc.     

CD97 neutralisation increases resistance to collagen-induced arthritis in mice

Synovial tissue of rheumatoid arthritis (RA) patients is characterised by an influx and retention of CD97-positive inflammatory cells. The ligands of CD97, CD55, chondroitin sulfate B, and α5β1 (very late antigen [VLA]-5) are expressed abundantly in the synovial tissue predominantly on fibroblast-like synoviocytes, endothelium, and extracellular matrix. Based upon this expression pattern, we hypothesise CD97 expression to result in accumulation of inflammatory cells in the synovial tissue of RA patients. To determine the therapeutic effect of blocking CD97 in an animal model of RA, collagen-induced arthritis was induced in a total of 124 DBA/J1 mice. Treatment was started on day 21 (early disease) or on day 35 (longstanding disease) with the blocking hamster anti-mouse CD97 monoclonal antibody (mAb) 1B2, control hamster immunoglobulin, or NaCl, applied intraperitoneally three times a week. The paws were evaluated for clinical signs of arthritis and, in addition, examined by radiological and histological analysis. Mice receiving 0.5 mg CD97 mAb starting from day 21 had significantly less arthritis activity and hind paw swelling. Furthermore, joint damage and inflammation were reduced and granulocyte infiltration was decreased. When treatment was started on day 35, CD97 mAb treatment had similar effects, albeit less pronounced. The results support the notion that CD97 contributes to synovial inflammation and joint destruction in arthritis.     

Deployment of ACT antimalarials for treatment of malaria: challenges and opportunities

Background

In late 2002, the health authorities of Mozambique implemented sulphadoxine-pyrimethamine (SP)/amodiaquine (AQ) as first-line treatment against uncomplicated falciparum malaria. In 2004, this has been altered to SP/artesunate in line with WHO recommendations of using Artemisinin Combination Therapies (ACTs), despite the fact that all the neighbouring countries have abandoned SP-drug combinations due to high levels of SP drug resistance. In the study area, one year prior to the change to SP/AQ, SP alone was used to treat uncomplicated malaria cases. The study described here investigated the immediate impact of the change to SP on the frequency of SP and CQ resistance-related haplotypes in the Plasmodium falciparum genes Pfdhfr, Pfdhps and Pfcrt before and a year after the introduction of SP.

Methods

Samples were collected during two cross sectional surveys in early 2002 and 2003 involving 796 and 692 children one year or older and adults randomly selected living in Maciana, an area located in Manhiça district, Southern Mozambique. Out of these, 171 and 173 P. falciparum positive samples were randomly selected to measure the frequency of resistance- related haplotypes in Pfdhfr, Pfdhps and Pfcrt based on results obtained by nested PCR followed by sequence-specific oligonucleotide probe (SSOP)-ELISA.

Results

The frequency of the SP-resistance associated Pfdhps double mutant (SGEAA) haplotype increased significantly from 14% to 35% (P < 0.001), while the triple mutant Pfdhfr haplotype (CIRNI) remained high and only changed marginally from 46% to 53% (P = 0.405) after one year with SP as first-line treatment in the study area. Conversely, the combined Pfdhfr/Pfdhps quintuple mutant haplotype increased from 8% to 26% (P = 0.005). The frequency of the chloroquine resistance associated Pfcrt -CVIET haplotype was above 90% in both years.

Conclusion

These retrospective findings add to the general concern on the lifespan of the combination of SP/artesunate in Mozambique. The high frequency of quintuple Pfdhfr/Pfdhps haplotypes found here as early as 2002 most likely cause ideal conditions for the development of artesunate tolerance in the P. falciparum populations and may even endanger the sensitivity to the second-line drug, Coartem^®.