Antagonism of Nodal signaling by BMP/Smad5 prevents ectopic primitive streak formation in the mouse amnion

The strength and spatiotemporal activity of Nodal signaling is tightly controlled in early implantation mouse embryos, including by autoregulation and feedback loops, and involves secreted and intracellular antagonists. These control mechanisms, which are established at the extra-embryonic/embryonic interfaces, are essential for anterior-posterior patterning of the epiblast and correct positioning of the primitive streak. Formation of an ectopic primitive streak, or streak expansion, has previously been reported in mutants lacking antagonists that target Nodal signaling. Here, we demonstrate that loss-of-function of a major bone morphogenetic protein (BMP) effector, Smad5, results in formation of an ectopic primitive streak-like structure in mutant amnion accompanied by ectopic Nodal expression. This suggests that BMP/Smad5 signaling contributes to negative regulation of Nodal. In cultured cells, we find that BMP-activated Smad5 antagonizes Nodal signaling by interfering with the Nodal-Smad2/4-Foxh1 autoregulatory pathway through the formation of an unusual BMP4-induced Smad complex containing Smad2 and Smad5. Quantitative expression analysis supports that ectopic Nodal expression in the Smad5 mutant amnion is induced by the Nodal autoregulatory loop and a slow positive-feedback loop. The latter involves BMP4 signaling and also induction of ectopic Wnt3. Ectopic activation of these Nodal feedback loops in the Smad5 mutant amnion results in the eventual formation of an ectopic primitive streak-like structure. We conclude that antagonism of Nodal signaling by BMP/Smad5 signaling prevents primitive streak formation in the amnion of normal mouse embryos.     

Einfluß einiger Faktoren in Abhängigkeit von den Methoden bei der Immobilisierung eines bakteriellen Enzympräparats mit Milchkoagulationswirkung

The influence of different factors (binding time, pH, temperature, and enzyme/carrier ratio) on the efficiency of immobilization of enzyme preparation with milk clotting activity from Bacillus mesentericus M strain by use of different carriers and methods of binding was investigated. It has been established that the dependence of immobilization from pH is most pronounced and is mainly influenced by the nature of the carrier. The binding time is of essential importance only by immobilization through adsorption. The temperature has not any influence on the efficiency of immobilization of the indicate preparation. Saturation of many of the used carriers by the different methods for immobilization was achieved at enzyme/carrier ratio 1 : 10, with exception of the cases where glutaraldehyde was used as binding reagent. In this case then the ratio was 1 : 4.     

LDLs induce fibroblast spreading independently of the LDL receptor via activation of the p38 MAPK pathway

Because adventitial fibroblasts play an important role in the repair of blood vessels, we assessed whether elevation in LDL concentrations would affect fibroblast function and whether this depended on activation of intracellular signaling pathways. We show here that in primary human fibroblasts, LDLs induced transient activation of the p38 mitogen-activated protein kinase (MAPK) pathway, but not the c-Jun N-terminal kinase MAPK pathway. This activation did not require the recruitment of the LDL receptor (LDLR), because LDLs efficiently stimulated the p38 MAPK pathway in human and mouse fibroblasts lacking functional LDLR, and because receptor-associated protein, an LDLR family antagonist, did not block the LDL-induced p38 activation. LDL particles also induced lamellipodia formation and cell spreading. These effects were blocked by SB203580, a specific p38 inhibitor. Our data demonstrate that LDLs can regulate the shape of fibroblasts in a p38 MAPK-dependent manner, a mechanism that may participate in wound healing or vessel remodeling as in atherosclerosis.     

Effect of temperature on some characteristics of the thermostable α-amylase from Bacillus licheniformis

The V _max of an extracellular, thermostable -amylase from Bacillus licheniformis 44MB82 were 5.70×10^-3 and 9.70×10^-3 mM s^-1 at 30 and 90°C, respectively, whereas the K _m values were similar (0.9 mg ml^-1) at both temperatures. Excluding dextrins, the dominant products from soluble starch and amylopectin hydrolysis contained less than six glucose residues. The enzyme hydrolysed amylopectin better than soluble starch. Increasing the temperature from 30 to 90°C was accompanied by an increase in the production of malto-oligosaccharides, especially maltotetrose, and this was related to the secondary hydrolysis of maltopentose and maltohexose.The authors are with the Institute of Microbiology, Bulgarian Academy of Sciences, Sofia 1113. 26 Academician G. Bonchev, Bulgaria     

Nitrile degradation by free and immobilized cells of the thermophile Bacillus sp. UG-5B, isolated from polluted industrial waters

A moderately thermophilic Gram-positive, sporulating, rod-shaped strain of Bacillus with nitrile-degrading activity was isolated from polluted industrial waters. Whole cells and cell-free extracts from the end of exponential growth phase expressed 7.6 nkat mg⁻¹ and 2.0 nkat mg⁻¹ benzonitrile-degrading activity, respectively, after cultivation in a fermentor with complex medium containing benzonitrile as an inducer. The benzonitrile degradation took place via the nitrilase pathway directly to benzoic acid without intermediate formation of benzamide. Samples with benzonitrilase activity of 7.6 nkat mg⁻¹ converted 3 mg benzonitrile in 1 h at 45°C. The half-life of benzonitrilase activity for a whole cell suspension and for cells immobilized in 2% agar was 4.5 min and 6 min at 70°C without substrate and 3 min at 90°C with substrate, respectively. The nitrilase had a broad substrate spectrum. The active biocatalyst obtained by immobilization was used in a continuous process and total biodegradation of 14.1 mM benzonitrile and 37.2 mM 4-cyanopyridine in a column bioreactor at 50°C for 5 h was achieved.     

Submerged culture process for biomass and exopolysaccharide production by Antarctic yeast: some engineering considerations

Production of biomass and extracellular polysaccharide (EPS) from psychrophilic Sporobolomyces salmonicolor AL₁ in a stirred bioreactor was studied. The aspects of production technical-scale parameters, namely, bioreactor flow field, biomass and EPS production rates, oxygen mass transfer per input power, as well as important product properties, such as rheology and stability of EPS mixtures, were considered. The bioprocess was found to proceed in non-Newtonian flow with consistency coefficient rising typically to 0.03 Pa.sⁿ and flow index declining to 0.7. Flow modeling was carried out and showed good homogenization for substrate delivery at agitation rates exceeding 400 rpm. Agitation rates lower than 400 rpm were considered counterproductive due to flow field non-uniformity. The cell density reached 5 g/l and EPS production yield reached 5.5 g/l at production rate 0.057 g EPS/l per hour (0.01 g EPS/g biomass per hour). Oxygen uptake rate and oxygen transfer rate were in the range of 0.5–1.7 mmolO₂/l per hour and 2–4.7 mmolO₂/l per hour, respectively. The mass transfer coefficient at reaction conditions was found to be in the range $ {K_L}a\tilde{\mkern6mu} 0.004-0.01{{\mathrm{s}}^{-1 }} $ . The bioprocess biological performance was higher at moderate agitation speed and revealed biomass diminution and cell inactivation by increasing impeller revolutions and shear rate. The product EPS was found to introduce shear-thinning behavior in water solutions with apparent viscosity of up to 30 mPa.s and to stabilize 1–2 % oil-in-water emulsions improving their lipophilic properties. The emulsion dispersion index was found to be comparable with the one of Arlacel 165, the emulsifier used in cosmetic. The long-term performance of the complex cream mixtures of the glucomannan prepared in commercial format was found promising for further application.