Thermoanaerobacter ethanolicus gen. nov., spec. nov., a new,extreme thermophilic,anaerobic bacterium

Two strains, JW 200 and JW 201, of an extreme thermophilic, non-spore-forming anaerobic bacterium were isolated from alkaline and slightly acidic hot springs located in Yellowstone National Park. Both strains were peritrichously flagellated rods. Cell size varied from 0.5–0.8 by 4–100 m; coccoid-shaped cells of about 1 m in diameter frequently occurred. Division was often unequal. Spheroplast-like forms were visible at the late logarithmic growth phase. The Gram reaction was variable. The DNA base composition of the two strains was between 37 and 39 mol% guanine plus cytosine as determined by buoyant density measurements and approximately 32% by the thermal denaturation method. The main fermentation products from hexoses were ethanol and CO₂. Growth occurred between 37 and 78°C and from pH 4.4 to 9.8. The name Thermoanaerobacter ethanolicus gen. nov., spec. nov. was proposed for the two, new isolates. Strain JW 200 was designated as the type strain.A preliminary account of this work was presented at the annual meeting of the American Society for Microbiology, Los Angeles, CA, 1979 (J. Wiegel and L. G. Ljungdahl, Abstr. Annu. Meet. Am. Soc. Microbiol., 1979, 163, p. 105) and at the 27th IUPAC Congress Helsinki, 1979 (L. G. Ljungdahl and J. Wiegel, Abstracts p. 546)     

Involvement of Mycobacterium smegmatis undecaprenyl phosphokinase in biofilm and smegma formation

We describe a Mycobacterium smegmatis mutant with impaired biofilm and smegma formation. A gene homologous to Escherichia coli bacA, which has been proposed to play a role as undecaprenyl phosphokinase (Upk) was unmarked in-frame deleted from M. smegmatis. Though Upk is involved in cell wall synthesis, the surface of the mutant strain appeared virtually comparable to that of the wild type by electron microscopy. The absence of Upk influenced colony morphology and bacitracin resistance. The M. smegmatis Deltaupk mutant developed a biofilm characterized by scattered islands of bacteria distinct from the completely covered biofilm surface observed for wild-type bacteria. We further demonstrate biological consequences of upk deletion for smegma development in an in vivo model. These results suggest the upk gene to be essential in biofilm and smegma development.     

Relationships between bacterial productivity and organic carbon at a soil—stream interface

Microbial communities at soil-stream interfaces may be particularly important in regulating amounts and forms of nutrients that leave upland soils and enter stream ecosystems. While microbial communities are thought to be responsible for key nutrient transformations within near-stream sediments, there is relatively little mechanistic information on factors that control microbial activities in these areas. In this study, we examine the roles of dissolved organic carbon (DOC) vs. particulate organic carbon (POC) as potential controls on rates of bacterial productivity (measured as incorporation of [3H]thymidine into bacterial DNA) and amounts of bacterial biomass (measured as fatty acid yield) in sediments along a transect perpendicular to a soil–stream interface. We hypothesized that spatial patterns in bacterial productivity would vary in response to strong and persistent patterns in pore-water concentrations of DOC that were observed along a soil-stream transect throughout a 2-year period. Our results did not support the existence of such a link between pore-water DOC and bacterial productivity. In contrast, we found bacterial productivity and biomass were related to small-scale spatial variations in sediment POC on 3 of 4 sample dates. While our results indicate that total bacterial productivity in near-stream sediments is not consistently linked to spatial variations in pore-water DOC, it is likely that DOC and POC are not mutually exclusive and the relative contribution of DOC and POC to sedimentary microbes varies temporally and spatially in different riparian habitats. This revised version was published online in July 2006 with corrections to the Cover Date.     

Association between plasma antibody response and protection in rainbow trout Oncorhynchus mykiss immersion vaccinated against Yersinia ruckeri

A key hallmark of the vertebrate adaptive immune system is the generation of antigen-specific antibodies from B cells. Fish are the most primitive gnathostomes (jawed vertebrates) possessing an adaptive immune system. Vaccination of rainbow trout against enteric redmouth disease (ERM) by immersion in Yersinia ruckeri bacterin confers a high degree of protection to the fish. The immune mechanisms responsible for protection may comprise both cellular and humoral elements but the role of specific immunoglobulins in this system has been questioned and not previously described. The present study demonstrates significant increase in plasma antibody titers following immersion vaccination and significantly reduced mortality during Y. ruckeri challenge.Rainbow trout were immersion-vaccinated, using either a commercial ERM vaccine (AquaVac™ ERM vet) or an experimental Y. ruckeri bacterin. Half of the trout vaccinated with AquaVac™ ERM vet received an oral booster (AquaVac™ ERM Oral vet). Sub-groups of the fish from each group were subsequently exposed to 1x10⁹ CFU Y. ruckeri/ml either eight or twenty-six weeks post vaccination (wpv). All vaccinated groups showed 0% mortality when challenged, which was highly significant compared to the non-vaccinated controls (40 and 28% mortality eight and twenty-six weeks post vaccination (wpv), respectively) (P<0.0001). Plasma samples from all groups of vaccinated fish were taken 0, 4, 8, 12, 16 and 26 wpv. and Y. ruckeri specific IgM antibody levels were measured with ELISA. A significant increase in titers was recorded in vaccinated fish, which also showed a reduced bacteremia during challenge. In vitro plasma studies showed a significantly increased bactericidal effect of fresh plasma from vaccinated fish indicating that plasma proteins may play a role in protection of vaccinated rainbow trout.     

Mannan Binding Lectin (MBL) genotypes coding for high MBL serum levels are associated with rheumatoid factor negative rheumatoid arthritis in never smokers

Introduction  

Previous studies have provided inconsistent results on whether variants in the MBL2 gene, coding for the complement-activating mannan-binding lectin (MBL) protein, associate with rheumatoid arthritis (RA). We re-evaluated this in context of the main environmental and genetic risk factors (smoking, HLA-DRB1 'shared epitope' (SE), PTPN22*620W), which predispose to rheumatoid factor (RF) and/or anti-citrullinated-protein antibody (ACPA)-positive RA.     

Control of hypocotyl elongation in Arabidopsis thaliana by photoreceptor interaction

In order to test the interaction of different phytochromes and blue-light receptors, etiolated seedlings of wild-type Arabidopsis thaliana (L.) Heynh., a phytochrome (phy) B-overexpressor line (ABO), and the photoreceptor mutants phyA-201, phyB-5, hy4-2.23n, fha-1, phyA-201/phyB-5, and phyA-201/hy4-2.23n were exposed to red and far-red light pulses after various preirradiations. The responsiveness to the inductive red pulses is primarily mediated by phyB which is rather stable in its far-red-absorbing form as demonstrated by a very slow loss of reversibility. Without preirradiation the red pulses had an impact on hypocotyl elongation only in PHYA mutants but not in the wild type. This indicates a suppression of phyB function by the presence of phyA. Preirradiation with either far-red or blue light resulted in an inhibition of hypocotyl elongation by red pulses in the wild type. Responsiveness amplification by far-red light is mediated by phyA and disappears slowly in the dark. The extent of responsiveness amplification by blue light was identical in the wild type and in the absence of phyA, or the cryptochromes cryl (hy4-2.23n) or cry2 (fha-1). Therefore, we conclude that stimulation of phyB by blue light preirradiation is either mediated by an additional still-unidentified blue-light-absorbing pigment or that phyA, cry1 and cry2 substitute for each other completely. Both blue and red preirradiation established responsiveness to red pulses in phyA-201/phyB-5 double mutants. These results demonstrate that inhibition of hypocotyl elongation by red pulses is not only mediated by phyB but also by a phytochrome(s) other than phyA and phyB. Received: 21 July 1998 / Accepted: 7 December 1998     

Antibodies against alpha-synuclein reduce oligomerization in living cells

Recent research implicates soluble aggregated forms of α-synuclein as neurotoxic species with a central role in the pathogenesis of Parkinson's disease and related disorders. The pathway by which α-synuclein aggregates is believed to follow a step-wise pattern, in which dimers and smaller oligomers are initially formed. Here, we used H4 neuroglioma cells expressing α-synuclein fused to hemi:GFP constructs to study the effects of α-synuclein monoclonal antibodies on the early stages of aggregation, as quantified by Bimolecular Fluorescence Complementation assay. Widefield and confocal microscopy revealed that cells treated for 48 h with monoclonal antibodies internalized antibodies to various degrees. C-terminal and oligomer-selective α-synuclein antibodies reduced the extent of α-synuclein dimerization/oligomerization, as indicated by decreased GFP fluorescence signal. Furthermore, ELISA measurements on lysates and conditioned media from antibody treated cells displayed lower α-synuclein levels compared to untreated cells, suggesting increased protein turnover. Taken together, our results propose that extracellular administration of monoclonal antibodies can modify or inhibit early steps in the aggregation process of α-synuclein, thus providing further support for passive immunization against diseases with α-synuclein pathology.     

Antitumor compound testing in glioblastoma organotypic brain cultures

Glioblastoma multiforme (GBM) is the most common and most aggressive type of primary brain tumor. Identification of new therapeutic regimens is urgently needed. A major challenge remains the development of a relevant in vitro model system with the necessary capacity and flexibility to profile compounds. The authors have developed and characterized a 3D culture system of brain cells (brain Hi-Spot) where GBM-derived cells can be incorporated (GBM/brain Hi-Spot). Immuno-fluorescence and electrophysiological recordings demonstrate that brain Hi-Spots recapitulate many features of brain tissue. Within this tissue, GBM-derived cell growth is monitored using a fluorescence assay. GBM-derived cells growing in Hi-Spots form tumor nodules that display properties of GBM such as 5-Ala positive staining, an acidic environment, and tumor-surrounding astrocyte activation. Temozolomide inhibits GBM growth in brain Hi-Spots, but it is not effective in 2D cultures. Other chemotherapeutics that have proven to be inefficient in GBM treatment display low activity against GBM-derived cells growing in brain Hi-Spots in comparison to their activity against GBM 2D cultures. These findings suggest that GBM/brain Hi-Spots represent a simple system to culture cells derived from brain tumors in an orthotopic environment in vitro and that the system is reliable to test GBM targeting compounds.     

Pig transgenesis by Sleeping Beauty DNA transposition

Modelling of human disease in genetically engineered pigs provides unique possibilities in biomedical research and in studies of disease intervention. Establishment of methodologies that allow efficient gene insertion by non-viral gene carriers is an important step towards development of new disease models. In this report, we present transgenic pigs created by Sleeping Beauty DNA transposition in primary porcine fibroblasts in combination with somatic cell nuclear transfer by handmade cloning. Göttingen minipigs expressing green fluorescent protein are produced by transgenesis with DNA transposon vectors carrying the transgene driven by the human ubiquitin C promoter. These animals carry multiple copies (from 8 to 13) of the transgene and show systemic transgene expression. Transgene-expressing pigs carry both transposase-catalyzed insertions and at least one copy of randomly inserted plasmid DNA. Our findings illustrate critical issues related to DNA transposon-directed transgenesis, including coincidental plasmid insertion and relatively low Sleeping Beauty transposition activity in porcine fibroblasts, but also provide a platform for future development of porcine disease models using the Sleeping Beauty gene insertion technology.