Methanogenic communities in permafrost-affected soils of the Laptev Sea coast, Siberian Arctic, characterized by 16S rRNA gene fingerprints

Permafrost environments in the Arctic are characterized by extreme environmental conditions that demand a specific resistance from microorganisms to enable them to survive. In order to understand the carbon dynamics in the climate-sensitive Arctic permafrost environments, the activity and diversity of methanogenic communities were studied in three different permafrost soils of the Siberian Laptev Sea coast. The effect of temperature and the availability of methanogenic substrates on CH4 production was analysed. In addition, the diversity of methanogens was analysed by PCR with specific methanogenic primers and by denaturing gradient gel electrophoresis (DGGE) followed by sequencing of DGGE bands reamplified from the gel. Our results demonstrated methanogenesis with a distinct vertical profile in each investigated permafrost soil. The soils on Samoylov Island showed at least two optima of CH4 production activity, which indicated a shift in the methanogenic community from mesophilic to psychrotolerant methanogens with increasing soil depth. Furthermore, it was shown that CH4 production in permafrost soils is substrate-limited, although these soils are characterized by the accumulation of organic matter. Sequence analyses revealed a distinct diversity of methanogenic archaea affiliated to Methanomicrobiaceae, Methanosarcinaceae and Methanosaetaceae. However, a relationship between the activity and diversity of methanogens in permafrost soils could not be shown.     

Ethidium monoazide and propidium monoazide for elimination of unspecific DNA background in quantitative universal real-time PCR

Unspecific background DNA in quantitative universal real-time PCR utilizing a hydrolysis probe was completely suppressed by the addition of EMA or PMA to the PCR mix via cross-linking of the dyes to DNA during 650 W visible light exposure. The proposed procedure had no effect on the sensitivity of the real-time PCR reaction.     

Antioxidant activity of isocoumarins isolated from Paepalanthus bromelioides on mitochondria

The isocoumarins (1-50 microM) paepalantine (9,10-dihydroxy-5,7-dimethoxy-1H-naptho(2,3c)pyran-1-one), 8,8'-paepalantine dimer, and vioxanthin isolated from Paepalanthus bromelioides, were assessed for antioxidant activity using isolated rat liver mitochondria and non-mitochondrial systems, and compared with the flavonoid quercetin. The paepalantine and paepalantine dimers, but not vioxanthin, were effective at scavenging both 1,1-diphenyl-2-picrylhydrazyl (DPPH(*)) and superoxide (O(2)(-)) radicals in non-mitochondrial systems, and protected mitochondria from tert-butylhydroperoxide-induced H(2)O(2) accumulation and Fe(2+)-citrate-mediated mitochondrial membrane lipid peroxidation, with almost the same potency as quercetin. These results point towards paepalantine, followed by paepalantine dimer, as being a powerful agent affording protection, apparently via O(2)(-) scavenging, from oxidative stress conditions imposed on mitochondria, the main intracellular source and target of those reactive oxygen species. This strong antioxidant action of paepalantine was reproduced in HepG2 cells exposed to oxidative stress condition induced by H(2)O(2).     

Impacts of T-Phylloplanin gene knockdown and of Helianthus and Datura phylloplanins on Peronospora tabacina spore germination and disease potential

T-phylloplanin proteins secreted to aerial surfaces of tobacco (Nicotiana tabacum) by short procumbent trichomes inhibit spore germination and blue mold disease caused by the oomycete pathogen Peronospora tabacina. Many other plants were found to contain water-washed leaf surface proteins (phylloplanins), but the functions and properties of these are not known. Here we extend earlier evidence for the antifungal activity of T-phylloplanins using a reverse genetics approach. RNA interference of the T-phylloplanin gene in tobacco 'T.I. 1068' resulted in loss of T-phylloplanin mRNA and protein, loss of in vitro spore germination inhibition activity, and leaf infection inhibition activity of leaf water washes from RNA interference plants, and young knockdown plants were susceptible to disease. The glycoprotein character, adaxial-leaf-surface enrichment of, and renewability of T-phylloplanins are also described. We also report that leaf water washes of sunflower (Helianthus annuus) and jimson weed (Datura metel), but not soybean (Glycine max), like that of tobacco, possess ProteinaseK- and boiling-sensitive P. tabacina spore germination and tobacco leaf infection inhibition activities. Results establish that T-phylloplaninins of tobacco are active in P. tabacina inhibition, and indicate that leaf surface proteins of certain non-Nicotiana species that are not susceptible to P. tabacina disease can inhibit germination of spores of this oomycete pathogen and inhibit tobacco leaf infection by this pathogen.     

Isolation of a field strain of Babesia bigemina (Piroplasma: Babesiidae) and establishment of in vitro culture for antigen production

Isolation of a field strain of Babesia bigemina (Piroplasma: Babesiidae) and establishment of in vitro culture for antigen production. Bovine b abesiosis, caused by Babesia bigemina, is a barrier for livestock development; it results in high economic loss to Mexican livestock. Control requires adequate antigens for diagnosis and vaccination programs. However, because of antigenic variation among Babesia strains, it is necessary to use antigens prepared from local strains. The purpose of the present study was to isolate a local field strain and to establish the in vitro culture of B. bigemina by the evaluation of the constituent's concentration of culture media. Thirty engorged female Boophilus microplus were collected from cattle suffering clinical babesiosis (B. bigemina) in Yucatan state, Mexico. These ticks were sent to the laboratory for detection of Babesia sp. vermicules. Eggs were kept at 83-85 % humidity and 27 degrees C until hatching. Larvae were transferred to an esplenectomized calf (B-1). The resulting nymphs were transferred to an esplenectomized calf (B-2). Twelve days later, B. bigemina (local strain) was detected in calf B-2 and its infected blood was frozen in liquid nitrogen to initiate the in vitro culture. The Microaerophilus Stationary Phase (MASP) in vitro culture method was used to reactivate the parasite. Three different concentrations of culture media (70, 60 and 50%), serum (30, 40 and 50%) and uninfected red blood cells (5, 10 and 15 %) were used in order to know the convenient concentrations to obtain the highest percentage of infected red blood cells (PEI). The cultured strain was used to prepare antigens for the Immunofluorescence Antibody Test (IFAT) and several concentrations of serum and conjugate were tested. Strain isolation was successful; 30 days were needed to obtain a PEI of 1.5%. The isolated strain was frozen in liquid nitrogen and the parasites were reactivated with the in vitro culture MASP method. The concentration of culture media that produced the highest PEI (14%) (p < 0.05) was 30% serum, 70% M199 and 5%. Uninfected Red Blood cells antigens were successfully used in the IFAT and the best dilutions to differentiate between positive and negative controls were serum 1:80 and conjugate 1:80. The isolated B. bigemina local strain requires particular conditions of in vitro culture by the MASP method to reach high numbers of infected red blood cells, needed to prepare and provide high quality antigens for serological diagnosis of B. bigemina.     

Circulation of Campylobacter spp. in rhesus monkeys (Macaca mulatta) held in captivity: a longitudinal study

Campylobacteriosis is an extremely important zoonosis, circulating freely in the environment. In nonhuman primates kept in open facilities and bred for experimental purposes, the presence of Campylobacter spp. could cause severe damage to the production and interfere with the results of scientific research. In this paper, we assessed the circulation of Campylobacter spp. in a colony of clinically healthy rhesus monkeys (Macaca mulatta) destined to research. The analysis was carried out during seven non-consecutive years. Data showed that despite several changes made in animal management along the studied years in order to control this zoonosis, reduction of bacterial charge did not occur. Significant differences among the age groups and sex were observed. Infants showed higher susceptibility than adult animals. In general males were more infected than females. Modifications adopted in the handling techniques need to be reviewed with the intent of improving the production, reducing bacterial infection of the stock and avoiding undesirable cross reactions in the research carried out with these animals. Therefore, this paper alerts professionals that work directly with captive rhesus monkeys about the risks of Campylobacter spp. infection and possible interference on the experimental procedures.     

Luminal antigens access late endosomes of intestinal epithelial cells enriched in MHC I and MHC II molecules: in vivo study in Crohn's ileitis

In contrast to healthy conditions, intestinal epithelial cells (IECs) stimulate proinflammatory CD4+ and CD8+ T cells during Crohn's disease (CD). The underlying regulatory mechanisms remain unknown. Here we investigated the epithelial expression of major histocompatibility complex (MHC) I and MHC II and its interference with endocytic pathways, in vivo. During ileoscopy, ovalbumin (OVA) was sprayed onto ileal mucosa of CD patients (ileitis and remission) and controls. The epithelial traffic of OVA and MHC I/II pathways were studied in biopsies using fluorescence and electron microscopy. We found MHC I and MHC II to accumulate within multivesicular late endosomes (MVLE) of IECs. Faint labeling for these molecules was seen in early endosomes and lysosomes. MVLE were entered by OVA 10 min after exposure. Exosomes carrying MHC I, MHC II, and OVA were detected in intercellular spaces of the epithelium. OVA trafficking and labeling patterns for MHC I and MHC II in IECs showed no differences between CD patients and controls. Independent of inflammatory stimuli, MHC I and MHC II pathways intersect MVLE in IECs, which were efficiently targeted by luminal antigens. Similar to MHC II-enriched compartments in professional antigen presenting cells, these MVLE might be critically involved in MHC I- and MHC II-related antigen processing in IECs and the source of epithelial-released exosomes. The access of luminal antigens to MHC I in MVLE might indicate that the presentation of exogenous antigens by IECs must not be restricted to MHC II but might also occur as "cross-presentation" via MHC I to CD8+ T cells.     

Cross-regulation of carbon monoxide and the adenosine A2a receptor in macrophages

Adenosine and heme oxygenase-1 (HO-1) exert a wide range of anti-inflammatory and immunomodulatory actions, making them crucial regulatory molecules. Despite the diversity in their modes of action, the similarity of biological effects of adenosine and HO-1 led us to hypothesize a possible interrelationship between them. We assessed a potential role for HO-1 in the ability of adenosine or 5'-N-ethylcarboxamidoadenosine (NECA), a stable adenosine analog, to modify the response of LPS-stimulated macrophages. Adenosine and NECA markedly induced HO-1 and blocked LPS-induced TNF-alpha production via adenosine A2aR-mediated signaling; blocking of HO-1 by RNA interference abrogated the effects of adenosine and NECA on TNF-alpha. HO-1 overexpression or exposure to carbon monoxide (CO), a product of HO-1 enzymatic activity, resulted in augmented A2aR mRNA and protein levels in RAW264.7 cells and primary macrophages. The induction of A2aR expression by HO-1 or CO resulted in an increase in the sensitivity to the anti-inflammatory effects of adenosine and NECA, which was lost in macrophages isolated from A2aR-deficient mice. Moreover, a decrease in cAMP levels upon NECA stimulation of naive macrophages was counterbalanced by CO exposure to up-regulate A2aR levels. This implies adenosine receptor isoform switch as a selective modification in macrophage phenotype. Taken together, these data suggest the existence of a positive feedback loop among adenosine, HO-1, CO, and the A2aR in the chronological resolution of the inflammatory response.