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Bacterial Production (bacterial + production)
Selected AbstractsEvolution of Amber Suppressor tRNAs for Efficient Bacterial Production of Proteins Containing Nonnatural Amino Acids,ANGEWANDTE CHEMIE, Issue 48 2009Jiantao Guo Angewandte Evolution: Bereiche der M.-jannaschii -Tyrosyl-tRNACUA, von denen man annimmt, dass sie mit dem Elongationsfaktor Tu wechselwirken, wurden randomisiert und die erhaltenen tRNA-Bibliotheken der In-vitro-Evolution unterworfen. Die dabei identifizierten tRNAs lieferten Proteine mit nichtnatürlichen Aminosäuren in deutlich höheren Ausbeuten. Manchmal hing das Ausmaß der Verbesserung von der Aminosäure ab. [source] Factors influencing bacterial dynamics along a transect from supraglacial runoff to proglacial lakes of a high Arctic glacieriFEMS MICROBIOLOGY ECOLOGY, Issue 2 2007Birgit Mindl Abstract Bacterial production in glacial runoff and aquatic habitats along a c. 500 m transect from the ablation area of a Svalbard glacier (Midre Lovénbreen, 79°N, 12°E) down to a series of proglacial lakes in its forefield were assessed. In addition, a series of in situ experiments were conducted to test how different nutrient sources (glacial flour and dissolved organic matter derived from goose faeces) and temperature affect bacterial abundance and production in these ecosystems. Bacterial abundance and production increased significantly along this transect and reached a maximum in the proglacial lakes. Bacterial diversity profiles as assessed by denaturing gradient gel electrophoresis indicated that communities in glacial runoff were different from those in proglacial lakes. Heterotrophic bacterial production was mainly controlled by temperature and phosphorus limitation. Addition of both glacial flour and dissolved organic matter derived from goose faeces stimulated bacterial production in those lakes. The results suggest that glacial runoff sustains an active bacterial community which is further stimulated in proglacial lakes by higher temperatures and nutrient inputs from bird faeces. Thus, as in maritime temperate and Antarctic settings, bacterial communities developing in the recently deglaciated terrain of Svalbard receive important inputs of nutrients via faunal transfers from adjacent ecosystems. [source] Diversity and abundance of freshwater Actinobacteria along environmental gradients in the brackish northern Baltic SeaENVIRONMENTAL MICROBIOLOGY, Issue 8 2009Karin Holmfeldt Summary Actinobacteria are highly abundant in pelagic freshwater habitats and also occur in estuarine environments such as the Baltic Sea. Because of gradients in salinity and other environmental variables estuaries offer natural systems for examining factors that determine Actinobacteria distribution. We studied abundance and community structure of Bacteria and Actinobacteria along two transects in the northern Baltic Sea. Quantitative (CARD-FISH) and qualitative (DGGE and clone libraries) analyses of community composition were compared with environmental parameters. Actinobacteria accounted for 22,27% of all bacteria and the abundance changed with temperature. Analysis of 549 actinobacterial 16S rRNA sequences from four clone libraries revealed a dominance of the freshwater clusters acI and acIV, and two new subclusters (acI-B scB-5 and acIV-E) were assigned. Whereas acI was present at all stations, occurrence of acII and acIV differed between stations and was related to dissolved organic carbon (DOC) and chlorophyll a (Chl a) respectively. The prevalence of the acI-A and acI-B subclusters changed in relation to total phosphorus (Tot-P) and Chl a respectively. Community structure of Bacteria and Actinobacteria differed between the river station and all other stations, responding to differences in DOC, Chl a and bacterial production. In contrast, the composition of active Actinobacteria (analysis based on reversely transcribed RNA) changed in relation to salinity and Tot-P. Our study suggests an important ecological role of Actinobacteria in the brackish northern Baltic Sea. It highlights the need to address dynamics at the cluster or subcluster phylogenetic levels to gain insights into the factors regulating distribution and composition of Actinobacteria in aquatic environments. [source] Factors influencing bacterial dynamics along a transect from supraglacial runoff to proglacial lakes of a high Arctic glacieriFEMS MICROBIOLOGY ECOLOGY, Issue 2 2007Birgit Mindl Abstract Bacterial production in glacial runoff and aquatic habitats along a c. 500 m transect from the ablation area of a Svalbard glacier (Midre Lovénbreen, 79°N, 12°E) down to a series of proglacial lakes in its forefield were assessed. In addition, a series of in situ experiments were conducted to test how different nutrient sources (glacial flour and dissolved organic matter derived from goose faeces) and temperature affect bacterial abundance and production in these ecosystems. Bacterial abundance and production increased significantly along this transect and reached a maximum in the proglacial lakes. Bacterial diversity profiles as assessed by denaturing gradient gel electrophoresis indicated that communities in glacial runoff were different from those in proglacial lakes. Heterotrophic bacterial production was mainly controlled by temperature and phosphorus limitation. Addition of both glacial flour and dissolved organic matter derived from goose faeces stimulated bacterial production in those lakes. The results suggest that glacial runoff sustains an active bacterial community which is further stimulated in proglacial lakes by higher temperatures and nutrient inputs from bird faeces. Thus, as in maritime temperate and Antarctic settings, bacterial communities developing in the recently deglaciated terrain of Svalbard receive important inputs of nutrients via faunal transfers from adjacent ecosystems. [source] Abundance and production of bacteria, and relationship to phytoplankton production, in a large tropical lake (Lake Tanganyika)FRESHWATER BIOLOGY, Issue 6 2009STEPHANE STENUITE Summary 1. Abundance and bacterial production (BP) of heterotrophic bacteria (HBact) were measured in the north and south basins of Lake Tanganyika, East Africa, during seasonal sampling series between 2002 and 2007. The major objective of the study was to assess whether BP can supplement phytoplankton particulate primary production (particulate PP) in the pelagic waters, and whether BP and particulate PP are related in this large lake. HBact were enumerated in the 0,100 m surface layer by epifluorescence microscopy and flow cytometry; BP was quantified using 3H-thymidine incorporation, usually in three mixolimnion layers (0,40, 40,60 and 60,100 m). 2. Flow cytometry allowed three subpopulations to be distinguished: low nucleic acid content bacteria (LNA), high nucleic acid content bacteria (HNA) and Synechococcus -like picocyanobacteria (PCya). The proportion of HNA was on average 67% of total bacterial abundance, and tended to increase with depth. HBact abundance was between 1.2 × 105 and 4.8 × 106 cells mL,1, and was maximal in the 0,40 m layer (i.e. roughly, the euphotic layer). Using a single conversion factor of 15 fg C cell,1, estimated from biovolume measurements, average HBact biomass (integrated over a 100-m water column depth) was 1.89 ± 1.05 g C m,2. 3. Significant differences in BP appeared between seasons, especially in the south basin. The range of BP integrated over the 0,100 m layer was 93,735 mg C m,2 day,1, and overlapped with the range of particulate PP (150,1687 mg C m,2 day,1) measured in the same period of time at the same sites. 4. Depth-integrated BP was significantly correlated to particulate PP and chlorophyll- a, and BP in the euphotic layer was on average 25% of PP. 5. These results suggest that HBact contribute substantially to the particulate organic carbon available to consumers in Lake Tanganyika, and that BP may be sustained by phytoplankton-derived organic carbon in the pelagic waters. [source] Lability of organic carbon in lakes of different trophic statusFRESHWATER BIOLOGY, Issue 6 2009A. P. OSTAPENIA Summary 1. We used first-order kinetic parameters of biological oxygen demand (BOD), the constant of aerobic decomposition (k) and the asymptotic value of BOD (BODult), to characterise the lability of organic carbon pools in six lakes of different trophic state: L. Naroch, L. Miastro and L. Batorino (Belarus), L. Kinneret (Israel), L. Ladoga (Russia) and L. Mendota (U.S.A.). The relative contributions of labile and refractory organic carbon fractions to the pool of total organic carbon (TOC) in these lakes were quantified. We also determined the amounts of labile organic carbon within the dissolved and particulate TOC pools in the three Belarus lakes. 2. Mean annual chlorophyll concentrations (used as a proxy for lake trophic state) ranged from 2.3 to 50.6 ,g L,1, labile organic carbon (OCL = 0.3BODult) from 0.75 to 2.95 mg C L,1 and k from 0.044 to 0.14 day,1. 3. Our data showed that there were greater concentrations of OCL but lower k values in more productive lakes. 4. In all cases, the DOC fraction dominated the TOC pool. OCL was a minor component of the TOC pool averaging about 20%, irrespective of lake trophic state. 5. In all the lakes, most (c. 85%) of the DOC pool was refractory, corresponding with published data based on measurements of bacterial production and DOC depletion. In contrast, a larger fraction (27,55%) of the particulate organic carbon (POC) pool was labile. The relative amount of POC in the TOC pool tended to increase with increasing lake productivity. 6. Long-term BOD incubations can be valuable in quantifying the rates of breakdown of the combined particulate and dissolved organic carbon pools and in characterising the relative proportions of the labile and recalcitrant fractions of these pools. If verified from a larger number of lakes our results could have important general implications. [source] Bacterial metabolism in small temperate streams under contemporary and future climatesFRESHWATER BIOLOGY, Issue 12 2007KAJ SAND-JENSEN Summary 1. We examined the detailed temperature dependence (0,40 °C) of bacterial metabolism associated with fine sediment particles from three Danish lowland streams to test if temperature dependence varied between sites, seasons and quality of organic matter and to evaluate possible consequences of global warming. 2. A modified Arrhenius model with reversible denaturation at high temperatures could account for the temperature dependence of bacterial metabolism and the beginning of saturation above 35 °C and it was superior to the unmodified Arrhenius model. Both models overestimated respiration rates at very low temperatures (<5 °C), whereas Ratkowsky's model , the square root of respiration , provided an excellent linear fit between 0 and 30 °C. 3. There were no indications of differences in temperature dependence among samples dominated by slowly or easily degradable organic substrates. Optimum temperature, apparent minimum temperature, Q10 -values for 0,40 °C and activation energies of bacterial respiration were independent of season, stream site and degradability of organic matter. 4. Q10 -values of bacterial respiration declined significantly with temperature (e.g. 3.31 for 5,15 °C and 1.43 for 25,35 °C) and were independent of site and season. Q10 -values of bacterial production behaved similarly, but were significantly lower than Q10 -values of respiration implying that bacterial growth efficiency declined with temperature. 5. A regional warming scenario for 2071,2100 (IPCC A2) predicted that mean annual temperatures will increase by 3.5 °C in the air and 2.2,4.3 °C in the streams compared with the control scenario for 1961,1990. Temperature is expected to rise more in cool groundwater-fed forest springs than in open, summer-warm streams. Mean annual bacterial respiration is estimated to increase by 26,63% and production by 18,41% among streams assuming that established metabolism,temperature relationships and organic substrate availability remain the same. To improve predictions of future ecosystem behaviour, we further require coupled models of temperature, hydrology, organic production and decomposition. [source] Seasonal and interannual variation of bacterial production in lowland rivers of the Orinoco basinFRESHWATER BIOLOGY, Issue 11 2004María M. Castillo Summary 1. We examined the influence of hydrologic seasonality on temporal variation of planktonic bacterial production (BP) in relatively undisturbed lowland rivers of the middle Orinoco basin, Venezuela. We sampled two clearwater and two blackwater rivers over 2 years for dissolved organic carbon (DOC), chlorophyll, phosphorus and bacterial abundance to determine their relationship to temporal variation in BP. 2. Dissolved organic carbon concentration was greater in blackwater (543,664 ,m) than in clearwater rivers (184,240 ,m), and was generally higher during periods of rising and high water compared with low water. Chlorophyll concentration peaked (3 ,g L,1) during the first year of study when discharge was lowest, particularly in blackwater rivers. Soluble reactive phosphorus (SRP) was very low in the study rivers (<3.8 ,g L,1) and concentration increased during low water. 3. Average BP was higher in clearwater (0.20,0.26 ,g C L,1 h,1) than in blackwater rivers (0.14,0.17 ,g C L,1 h,1), although mean bacterial abundance was similar among rivers (0.6,0.8 × 106 cells mL,1). 4. Periods of higher chlorophyll a concentration (low water) or flushing of terrestrial organic material (rising water) were accompanied by higher BP, while low BP was observed during the period of high water. 5. Interannual variation in BP was influenced by variations in discharge related to El Niño Southern Oscillation events. 6. Seasonal variation in BP in the study rivers and other tropical systems was relatively small compared with seasonal variation in temperate rivers and lakes. In addition to the low seasonal variation of temperature in the tropics, low overall human disturbance could result in less variation in the inputs of nutrients and carbon to the study rivers compared with more disturbed temperate systems. [source] Modulation of the bacterial response to spectral solar radiation by algae and limiting nutrientsFRESHWATER BIOLOGY, Issue 11 2002J. M. Medina-Sánchez SUMMARY 1. The response of bacterial production (measured as [3H]TdR incorporation rate) to spectral solar radiation was quantified experimentally in an oligotrophic high-mountain lake over 2 years. Bacterial responses were consistent: ultraviolet-B (UVB) was harmful, whereas ultraviolet-A (UVA) + photosynthetically active radiation (PAR) and PAR enhanced bacterial activity. Full sunlight exerted a net stimulatory effect on bacterial activity in mid-summer but a net inhibitory effect towards the end of the ice-free period. 2. Experiments were undertaken to examine whether the bacterial response pattern depended on the presence of algae and/or was modulated by the availability of a limiting inorganic nutrient (phosphorus, P). In the absence of algae, [3H]TdR incorporation rates were significantly lower than when algae were present under all light treatments, and the consistent bacterial response was lost. This suggests that the bacterial response to spectral solar radiation depends on fresh-C released from algae, which determines the net stimulatory outcome of damage and repair in mid-summer. 3. In the absence of algae, UVB radiation inhibited bacteria when they were strongly P-deficient (mean values of N : P ratio: 46.1), whereas it exerted no direct effect on bacterial activity when they were not P-limited. 4. P-enrichment of lake water markedly altered the response of bacteria to spectral solar radiation at the end of ice-free period, when bacteria were strongly P-deficient. Phosphorus enrichment suppressed the inhibitory effect of full sunlight that was observed in October, both in whole lake water (i.e. including algae) and in the absence of algae. This indicates that the bacterial P-deficiency, measured as the cellular N : P ratio, was partly responsible for the net inhibitory effect of full sunlight, implying a high bacterial vulnerability to UVB. [source] Effect of Campsurus notatus on NH+4, DOC Fluxes, O2 Uptake and Bacterioplankton Production in Experimental Microcosms with Sediment-Water Interface of an Amazonian Lake Impacted by Bauxite TailingsINTERNATIONAL REVIEW OF HYDROBIOLOGY, Issue 2 2003João José Fonseca Leal Abstract The aim of this study was to evaluate the influence of Campusurus notatusEaton 1868 (Ephemeroptera: Polimitarciydae) and the impact of bauxite tailings on ammonium (NH4+) and dissolved organic carbon (DOC) fluxes, oxygen uptake and bacterioplankton production in the sediment-water interface of Lake Batata, a shallow Amazonian floodplain lake. Mesocosms were constructed from natural and impacted areas of Lake Batata, to reproduce the sediment-water interface. The cores were incubated with 0 to 2,388 ind m,2 of Campsurus notatus nymphs, and the changes in NH4+, DOC, O2 concentration and bacterioplankton production in the overlying water column were measured. Ammonium efflux (F = 9.8, p < 0.05, multiple regression) and oxygen uptake (F = 11.8, p < 0.05) showed a significant correlation with the density of C. notatus in the cores with natural sediment. No differences on DOC release were observed in cores with natural or impacted sediment. In the cores incubated with natural sediment and nymphs of C. notatus, a significant increase (Two-way ANOVA, p < 0.05) in bacterial production (0.44 ,g C l,1 h,1) was observed after 3 hours of incubation. In cores incubated with sediment impacted by bauxite tailings, there was no difference in bacterial production with and without C. notatus. We conclude that C. notatus is an important bioturbator in Lake Batata, increasing the turnover rate of nitrogen (NH4+) at the sediment-water interface and bacterial production in cores incubated with natural sediment. It is also clear that bauxite tailings reduce the nutrients turnover rates in impacted regions of Lake Batata and influence bacterial production. [source] ORIGINAL ARTICLE: Application of soybean meal, soy protein concentrate and isolate differing in , -galactosides content to low- and high-fibre diets in growing turkeysJOURNAL OF ANIMAL PHYSIOLOGY AND NUTRITION, Issue 5 2010Z. Zdu, czyk Summary The aim of this experiment was to investigate the physiological and growth response of young turkeys (up to 8 weeks of age) to dietary replacement of soybean meal (SBM) by soy protein concentrate (PC) or protein isolate (PI). This replacement resulted in a differentiated dietary concentration of , -galactosides of over 2.5% in the SBM diet, approximately 2% with a mixture SBM and PC, 1% with a PC diet and 0.1% with a PI diet. Each treatment was applied in two ways: with lower (3.5%) or higher (5.3%) dietary crude fibre content, made by supplementation with soybean hulls. The highest and lowest body weight of turkeys was recorded both after the first and second 4-week half of the study in the PC and PI-type diets respectively. A gradual withdrawal of , -galactosides from a diet was accompanied by a decline in ileal tissue mass, ileal viscosity and activity of endogenous maltase (the latter was found to be significant at 4 weeks of age). At the same time, two-way anova revealed that an elevated level of crude fibre (HF treatment) caused an increase in ileal tissue mass (p < 0.05 after 4 weeks of feeding) as well as a decrease in activity level of intestinal sucrase and maltase. The presence of raffinose family oligosaccharides in a diet, in contrast to dietary crude fibre level, significantly affected the caecal metabolism. The rate of bacterial production of short-chain fatty acids in the caeca was distinctly diminished by dietary withdrawal of , -galactosides. In conclusion, the soy protein concentrate, in contrast to the protein isolate preparation, exerted positive effects on the turkeys' growth and gastrointestinal tract physiology and should be considered as an effective SBM substitute. [source] Unusual hypersensitivity to warfarin in a critically ill patientJOURNAL OF CLINICAL PHARMACY & THERAPEUTICS, Issue 5 2004H. Konishi PhD Summary A patient was admitted to the intensive care unit because of respiratory failure, and warfarin therapy was started at 2 mg/day for the treatment of pulmonary embolism, together with other medications. Despite the low dosage of warfarin, international normalized ratio (INR) was markedly elevated from 1·15 to 11·28 for only 4 days, and bleeding symptoms concurrently developed. Vitamin K2 was infused along with discontinuation of warfarin. One day later, the INR was found to have decreased, and bleeding was also improved. An objective causality assessment indicated a probable relationship between clotting abnormality and warfarin administration, although the degree of elevation of the INR was unusual in the light of the daily warfarin dose and duration of its exposure. Based on the clinical status of the patient, it was suspected that several conditions contributed to the abnormal hypersensitivity to warfarin. Contributory factors probably included pharmacokinetic interactions with co-administrated drugs, vitamin K deficiency caused by decreased dietary intake, reduced gut bacterial production, impaired intestinal absorption and hepatic synthetic capacity, and increased consumption of clotting factors. In view of our experience in the present case, it should be stressed that close monitoring of coagulation capacity is necessary in critically ill patients in order to avoid fatal haemorrhage after initiating warfarin therapy regardless of the dosage. [source] Seasonal and diel changes of dissolved oxygen in a hypertrophic prairie lakeLAKES & RESERVOIRS: RESEARCH AND MANAGEMENT, Issue 3 2005Richard D. Robarts Abstract Humboldt Lake, a hypertrophic prairie lake typical of many found on the Great Plains of North America, is usually ice-covered from early November to about mid-May. The lake is an important recreational fishery, now mainly stocked with walleye. It has a high potential risk of experiencing fish kills because of the very large cyanobacterial blooms that develop in it, the high rates of algal and bacterial production and the high concentrations of ammonia (NH3 -N) and dissolved organic matter. Following the collapse of cyanobacterial blooms, shallow prairie lakes are known to undergo periods of anoxia that can lead to summer fish kills. In some of the lakes, anoxia forms during the long period of ice cover, causing winter fish kills. Two years of seasonal and diel data (total phosphorus, dissolved oxygen (DO), NH3 -N and chlorophyll- a concentrations, and bacterial production) were analysed in this study to assess why significant fish kills did not occur during this period or during the , 30 years of records from Saskatchewan Environment. Humboldt Lake did not become anaerobic, either following the collapse of the cyanobacterial bloom or under ice cover, indicating that the oxygen (O2) influx (strong mixing) and production processes were greater than the microbial and chemical O2 demands, both over seasonal and diel time scales. Several published risk threshold criteria to predict the probability of summer and/or winter fish kills were applied in this study. The threshold criteria of maximum summer chlorophyll and maximum winter NH3 -N concentrations indicated that a summer fish kill was unlikely to occur in this hypertrophic prairie lake, provided its water quality remained similar to that during this study. Similarly, the threshold criteria of initial DO storage before ice cover and the rate of O2 depletion under ice cover also indicated a winter fish kill was unlikely. However, recent development in the watershed might have resulted in significant water quality deterioration and the winter fish kill that occurred in 2005. [source] Ultraviolet-B Radiation Effects on the Structure and Function of Lower Trophic Levels of the Marine Planktonic Food WebPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2006Gustavo A. Ferreyra ABSTRACT The impact of UV-B radiation (UVBR; 280,320 nm) on lower levels of a natural plankton assemblage (bacteria, phytoplankton and microzooplankton) from the St. Lawrence Estuary was studied during 9 days using several immersed outdoor mesocosms. Two exposure treatments were used in triplicate mesocosms: natural UVBR (N treatment, considered as the control treatment) and lamp-enhanced UVBR (H treatment, simulating 60% depletion of the ozone layer). A phytoplankton bloom developed after day 3, but no significant differences were found between treatments during the entire experiment for phytoplankton biomass (chlorophyll a and cell carbon) nor for phytoplankton cell abundances from flow cytometry and optical microscopy of three phytoplankton size classes (picoplankton, nanoplankton and microplankton). In contrast, bacterial abundances showed significantly higher values in the H treatment, attributed to a decrease in predation pressure due to a dramatic reduction in ciliate biomass (, 70,80%) in the H treatment relative to the N treatment. The most abundant ciliate species were Strombidinium sp., Prorodon ovum and Tintinnopsis sp.; all showed significantly lower abundances under the H treatment. P. ovum was the less-affected species (50% reduction in the H treatment compared with that of the N control), contrasting with ,90% for the other ones. Total specific phytoplanktonic and bacterial production were not affected by enhanced UVBR. However, both the ratio of primary to bacterial biomass and production decreased markedly under the H treatment. In contrast, the ratio of phytoplankton to bacterial plus ciliate carbon biomass showed an opposite trend than the previous results, with higher values in the H treatment at the end of the experiment. These results are explained by the changes in the ciliate biomass and suggest that UVBR can alter the structure of the lower levels of the planktonic community by selectively affecting key species. On the other hand, linearity between particulate organic carbon (POC) and estimated planktonic carbon was lost during the postbloom period in both treatments. On the basis of previous studies, our results can be attributed to the aggregation of carbon released by cells to the water column in the form of transparent exopolymer particles (TEPs) under nutrient limiting conditions. Unexpectedly, POC during such a period was higher in the H treatment than in controls. We hypothesize a decrease in the ingestion of TEPs by ciliates, in coincidence with increased DOC release by phytoplankton cells under enhanced UVBR. The consequences of such results for the carbon cycle in the ocean are discussed. [source] Identification of potential substrate proteins for the periplasmic Escherichia coli chaperone SkpPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 23-24 2008Svenja Jarchow Abstract The "seventeen kilodalton protein" (Skp) is a predominant periplasmic chaperone of Escherichia coli, which is involved in the biogenesis of abundant outer membrane proteins (OMPs) such as OmpA, PhoE, and LamB. In this study the substrate profile of Skp was investigated in a proteomics approach. Skp was overexpressed in a deficient E. coli strain as a fusion protein with the Strep,tag and captured, together with any host proteins associated with it, from the periplasmic cell extract under mild conditions via one-step Strep,Tactin affinity chromatography. Copurified substrate proteins were then identified by high resolution 2-DE with immobilized pH-gradients, followed by MALDI-TOF MS. Apart from the known Skp substrates, including OmpA and LamB, more than 30 other interacting proteins were detected, especially from the outer membrane, among these FadL and BtuB, and from the periplasm such as MalE and OppA. Thus, Skp does not only serve as a specialized chaperone for a small set of OMPs, but it seems to exhibit a broader substrate spectrum, including soluble periplasmic proteins. These findings should prompt further investigation into the physiological role of Skp and may promote its use for the bacterial production of biochemically active heterologous proteins whose folding requires secretion into the oxidizing milieu of the periplasm. [source] Formatted anti,tumor necrosis factor , VHH proteins derived from camelids show superior potency and targeting to inflamed joints in a murine model of collagen-induced arthritisARTHRITIS & RHEUMATISM, Issue 6 2006Ken Coppieters Objective The advent of tumor necrosis factor (TNF),blocking drugs has provided rheumatologists with an effective, but highly expensive, treatment for the management of established rheumatoid arthritis (RA). Our aim was to explore preclinically the application of camelid anti-TNF VHH proteins, which are single-domain antigen binding (VHH) proteins homologous to human immunoglobulin VH domains, as TNF antagonists in a mouse model of RA. Methods Llamas were immunized with human and mouse TNF, and antagonistic anti-TNF VHH proteins were isolated and cloned for bacterial production. The resulting anti-TNF VHH proteins were recombinantly linked to yield bivalent mouse and human TNF-specific molecules. To increase the serum half-life and targeting properties, an anti,serum albumin anti-TNF VHH domain was incorporated into the bivalent molecules. The TNF-neutralizing potential was analyzed in vitro. Mouse TNF-specific molecules were tested in a therapeutic protocol in murine collagen-induced arthritis (CIA). Disease progression was evaluated by clinical scoring and histologic evaluation. Targeting properties were evaluated by 99mTc labeling and gamma camera imaging. Results The bivalent molecules were up to 500 times more potent than the monovalent molecules. The antagonistic potency of the anti-human TNF VHH proteins exceeded even that of the anti-TNF antibodies infliximab and adalimumab that are used clinically in RA. Incorporation of binding affinity for albumin into the anti-TNF VHH protein significantly prolonged its serum half-life and promoted its targeting to inflamed joints in the murine CIA model of RA. This might explain the excellent therapeutic efficacy observed in vivo. Conclusion These data suggest that because of the flexibility of their format, camelid anti-TNF VHH proteins can be converted into potent therapeutic agents that can be produced and purified cost-effectively. [source] Genetic analysis of G protein-coupled receptor expression in Escherichia coli: Inhibitory role of DnaJ on the membrane integration of the human central cannabinoid receptorBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2009Georgios Skretas Abstract The overexpression of G protein-coupled receptors (GPCRs) and of many other heterologous membrane proteins in simple microbial hosts, such as the bacterium Escherichia coli, often results in protein mistargeting, aggregation into inclusion bodies or cytoplasmic degradation. Furthermore, membrane protein production is very frequently accompanied by severe cell toxicity. In this work, we have employed a genetic strategy to isolate E. coli mutants that produce markedly increased amounts of the human central cannabinoid receptor (CB1), a pharmacologically significant GPCR that expresses very poorly in wild-type E. coli. By utilizing a CB1 fusion with the green fluorescent protein (GFP) and fluorescence-activated cell sorting (FACS), we screened an E. coli transposon library and identified an insertion in dnaJ that resulted in a large increase in CB1-GFP fluorescence and a dramatic enhancement in bacterial production of membrane-integrated CB1. Furthermore, the dnaJ::Tn5 inactivation suppressed the severe cytotoxicity associated with CB1 production. This revealed an unexpected inhibitory role of the chaperone/ co-chaperone DnaJ in the protein folding or membrane insertion of bacterially produced CB1. Our strategy can be easily adapted to identify expression bottlenecks for different GPCRs or any other integral membrane protein, provide useful and unanticipated mechanistic insights, and assist in the construction of genetically engineered E. coli strains for efficient heterologous membrane protein production. Biotechnol. Bioeng. 2009;102: 357,367. © 2008 Wiley Periodicals, Inc. 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