Slow-release inoculation allows sustained biodegradation of gamma-hexachlorocyclohexane

This study investigated the feasibility of a slow-release inoculation approach as a bioaugmentation strategy for the degradation of lindane (gamma-hexachlorocyclohexane [gamma-HCH]). Slow-release inoculation of Sphingomonas sp. gamma 1-7 was established in both liquid and soil slurry microcosms using open-ended silicone tubes in which the bacteria are encapsulated in a protective nutrient-rich matrix. The capacity of the encapsulated cells to degrade lindane under aerobic conditions was evaluated in comparison with inoculation of free-living cells. Encapsulation of cells in tubes caused the removal of lindane by adsorption to the silicone tubes but also ensured prolonged biodegradation activity. Lindane degradation persisted 2.2 and 1.4 times longer for liquid and soil slurry microcosms, respectively, than that for inoculation with free cells. While inoculation of free-living cells led to a loss in lindane-degrading activity in limited time intervals, encapsulation in tubes allowed for a more stable actively degrading community. The loss in degrading activity was linked to the loss of the linA gene, encoding gamma-HCH dehydrochlorinase (LinA), which is involved in the initial steps of the lindane degradation pathway. This work shows that a slow-release inoculation approach using a catabolic strain encapsulated in open-ended tubes is a promising bioaugmentation tool for contaminated sites, as it can enhance pollutant removal and can prolong the degrading activity in comparison with traditional inoculation strategies.     

Exploring the Unique N-Glycome of the Opportunistic Human Pathogen Acanthamoeba

Glycans play key roles in host-pathogen interactions; thus, knowing the N-glycomic repertoire of a pathogen can be helpful in deciphering its methods of establishing and sustaining a disease. Therefore, we sought to elucidate the glycomic potential of the facultative amoebal parasite Acanthamoeba. This is the first study of its asparagine-linked glycans, for which we applied biochemical tools and various approaches of mass spectrometry. An initial glycomic screen of eight strains from five genotypes of this human pathogen suggested, in addition to the common eukaryotic oligomannose structures, the presence of pentose and deoxyhexose residues on their N-glycans. A more detailed analysis was performed on the N-glycans of a genotype T11 strain (4RE); fractionation by HPLC and tandem mass spectrometric analyses indicated the presence of a novel mannosylfucosyl modification of the reducing terminal core as well as phosphorylation of mannose residues, methylation of hexose and various forms of pentosylation. The largest N-glycan in the 4RE strain contained two N-acetylhexosamine, thirteen hexose, one fucose, one methyl, and two pentose residues; however, in this and most other strains analyzed, glycans with compositions of Hex_8–9HexNAc₂Pnt_0–1 tended to dominate in terms of abundance. Although no correlation between pathogenicity and N-glycan structure can be proposed, highly unusual structures in this facultative parasite can be found which are potential virulence factors or therapeutic targets.     

The complete genome sequence of Bacillus licheniformis DSM13, an organism with great industrial potential

The genome of Bacillus licheniformis DSM13 consists of a single chromosome that has a size of 4,222,748 base pairs. The average G+C ratio is 46.2%. 4,286 open reading frames, 72 tRNA genes, 7 rRNA operons and 20 transposase genes were identified. The genome shows a marked co-linearity with Bacillus subtilis but contains defined inserted regions that can be identified at the sequence as well as at the functional level. B. licheniformis DSM13 has a well-conserved secretory system, no polyketide biosynthesis, but is able to form the lipopeptide lichenysin. From the further analysis of the genome sequence, we identified conserved regulatory DNA motives, the occurrence of the glyoxylate bypass and the presence of anaerobic ribonucleotide reductase explaining that B. licheniformis is able to grow on acetate and 2,3-butanediol as well as anaerobically on glucose. Many new genes of potential interest for biotechnological applications were found in B. licheniformis; candidates include proteases, pectate lyases, lipases and various polysaccharide degrading enzymes.     

Local nitric oxide levels reflect the degree of allergic airway inflammation after segmental allergen challenge in asthmatics.

Nitric oxide (NO) levels are increased in the exhaled air of asthmatics. As NO levels correlate with allergic airway inflammation, NO measurement has been suggested for disease monitoring. In patients with asthma, we previously demonstrated that intrabronchial treatment with a natural porcine surfactant enhanced airway inflammation after segmental allergen provocation. We studied whether local levels of NO reflect the degree of allergic airway inflammation following segmental allergen challenge with or without surfactant pretreatment. Segmental NO, as well as nitrite and nitrate in bronchoalveolar lavage (BAL) fluid, was measured before and after segmental challenge with either saline, saline plus allergen, or surfactant plus allergen in 16 patients with asthma and five healthy subjects. The data were compared with inflammatory BAL cells. Segmental NO levels were increased after instillation of saline (p < 0.05), or surfactant plus allergen in asthmatics (p < 0.05), and values were higher after surfactant plus allergen compared to saline challenge. Nitrate BAL levels were not altered after saline challenge but increased after allergen challenge (p < 0.05) and further raised by surfactant (p < 0.05), whereas nitrite levels were not altered by any treatment. Segmental NO and nitrate levels correlated with the degree of eosinophilic airway inflammation, and nitrate levels also correlated with neutrophil and lymphocyte numbers in BAL. In healthy subjects, NO, nitrite, and nitrate were unaffected. Thus, segmental NO and nitrate levels reflect the degree of allergic airway inflammation in patients with asthma. Measurement of both markers can be useful in studies using segmental allergen provocation, to assess local effects of potential immunomodulators.     

Levels of tropinone-reductase activities influence the spectrum of tropane esters found in transformed root cultures of Datura stramonium L.

The nortropane sulphur analogues 8-thiabicyclo[3.2.1] octan-3-one, 8-thiabicyclo[3.2.1]octan-3a-ol and 8-thiabicyclo[3.2.1]octan-3-ol have been found to have differential effects in vitro on the activities of tropinone reductase I and tropinone reductase II from Datura stramonium L. It has been demonstrated that only tropinone reductase I is able to metabolise 8-thiabicyclo[3.2.1]octan-3-one and that only this enzyme is inhibited by 8-thiabicyclo[3.2.1]octan-3-ol and 8-thiabicyclo[3.2.1]octan-3-ol. A K _m of 0.035 mM was determined for 8-thiabicyclo[3.2.1]octan-3-one and I₅₀ values of 0.081 mM and 0.021 mM for 8-thiabicyclo[3.2.1]octan-3-ol and 8-thiabicyclo[3.2.1]octan-3-ol, respectively. The influence that these differential interactions might have on metabolism was investigated in transformed root cultures of D. stramonium. It was found that when these cultures were grown in the presence of either 8-thiabicyclo[3.2.1]octan-3-one or 8-thiabicyclo[3.2.1]octan-3-ol the spectrum of alkaloids that accumulated was altered from that found in control roots in the manner predicted from the observed effects of these inhibitors on the isolated reductases. The effect could be mimicked by feeding pseudotropine, the product of tropinone reductase II. It is concluded that the relative levels of activity of the two tropinone reductases might play an important role in regulating the balance of tropan-3-ols to tropan-3-ols seen in the spectrum of tropane-alkaloid-producing plants.Abbreviations GC/MS gas chromatography/mass spectrometry; - I₅₀ concentration of inhibitor required to reduce the rate of reaction to half the maximal value; - -TBOL 8-thiabicyclo[3.2.1]octan-3-ol; - -TBOL 8-thiabicyclo[3.2.1]octan-3-ol; - TBON 8-thiabicyclo[3.2.1]octan-3-one; - TR tropinone reductase We are most grateful to J. Eagles (I.F.R., Norwich) for GC/MS analysis, to colleagues at I.P.B.P. and I.F.R. for helpful discussions, to the technical staff (Chemistry, Glasgow) and to W. Millar (Chemistry, Glasgow) for assistance with the reduction of TBON. This work was, in part, supported by a grant to B Dräger from the Deutsche Forschungsgemeinschaft (Dr227/I-I). The research reported here was supported by an Academic Research Collaboration Cooperative Award (project No. 215) from the British Council and the Deutscher Akademischer Austauschdienst to R.J. Robins and B. Dräger.     

Regulation of gap junction coupling in the developing neocortex

In the developing mammalian, neocortex gap junctions represent a transient, metabolic, and electrical communication system. These gap junctions may play a crucial role during the formation and refinement of neocortical synaptic circuitries. This article focuses on two major points. First, the influence of gap junctions on electrotonic cell properties will be considered. Both the time-course and the amplitude of synaptic potentials depend,inter alia, on the integration capabilities of the postsynaptic neurons. These capabilities are, to a considerable extent, determined by the electrotonic characteristics of the postsynaptic cell. As a consequence, the efficacy of chemical synaptic inputs may be crucially affected by the presence of gap junctions. The second major topic is the regulation of gap junctional communication by neurotransmitters via second messenger pathways. The monoaminergic neuromodulators dopamine, nordrenaline, and serotonin reduce gap junction coupling via activation of two different intracellular signaling cascades—the cAMP/protein kinase A pathway and the IP₃/Ca²⁺/protein kinase C pathway, 013 respectively. In addition, gap junctional communication seems to be modulated by the nitric oxide (NO)/cGMP system. Since NO production can be stimulated by glutamate-induced calcium influx, the NO/cGMP-dependent modulation of gap junctions might represent a functional link between developing glutamatergic synaptic transmission and the gap junctional network. Thus, it might be of particular importance in view of a role of gap junctions during the process of circuit formation.     

Tumor necrosis factor and norepinephrine lower the levels of human neutrophil peptides 1-3 secretion by mixed synovial tissue cultures in osteoarthritis and rheumatoid arthritis

Introduction  

Neutrophils and monocytes play an important role in overt inflammation in chronic inflammatory joint diseases such as rheumatoid arthritis (RA). The sympathetic nervous system (SNS) inhibits many neutrophil/monocyte functions and macrophage tumor necrosis factor (TNF), but because of the loss of sympathetic nerve fibers in inflamed tissue, sympathetic control is attenuated. In this study, we focused on noradrenergic and TNF regulation of human neutrophil peptides 1-3 (HNP1-3), which are proinflammatory bactericidal α-defensins.     

Susceptibility Screening of Hyphae-Forming Fungi with a New, Easy, and Fast Inoculum Preparation Method

In vitro susceptibility testing of clinically important fungi becomes more and more essential due to the rising number of fungal infections in patients with impaired immune system. Existing standardized microbroth dilution methods for in vitro testing of molds (CLSI, EUCAST) are not intended for routine testing. These methods are very time-consuming and dependent on sporulating of hyphomycetes. In this multicentre study, a new (independent of sporulation) inoculum preparation method (containing a mixture of vegetative cells, hyphae, and conidia) was evaluated. Minimal inhibitory concentrations (MIC) of amphotericin B, posaconazole, and voriconazole of 180 molds were determined with two different culture media (YST and RPMI 1640) according to the DIN (Deutsches Institut für Normung) microdilution assay. 24 and 48?h MIC of quality control strains, tested per each test run, prepared with the new inoculum method were in the range of DIN. YST and RPMI 1640 media showed similar MIC distributions for all molds tested. MIC readings at 48 versus 24?h yield 1 log₂ higher MIC values and more than 90?% of the MICs read at 24 and 48?h were within ±2 log₂ dilution. MIC end point reading (log_{2 MIC-RPMI 1640}?log_{2 MIC-YST}) of both media demonstrated a tendency to slightly lower MICs with RPMI 1640 medium. This study reports the results of a new, time–saving, and easy-to-perform method for inoculum preparation for routine susceptibility testing that can be applied for all types of spore-/non-spore and hyphae-forming fungi.