Biochemical reactions of Brevibacterium flavum depending on medium stirring intensity and flow structure

Authors have studied the dependence of the physiological and biochemical characteristics of lysine producers Brevibacterium flavum on medium stirring intensity upon constant pO₂ during fermentation processes. Various factors affecting the rate of biochemical reactions in cells upon a changeable intensity of medium stirring in apparata with turbine stirrers; oxygen mass transfer intensity in the system; stirring characteristics in the fermenter; biochemical changes in the cells due to gradients of energy introduced by the stirrer in the fermenter, etc. have been studied. A comparison of the experimentally established values, characterizing macrostirring (e) and microstirring (K_R), with the physiological and chemical characteristics of bacteria during fermentation made us suppose that the character of the kinetic energy distribution in the volume of fermenter is one of the factors regulating bacterial metabolism. Besides, there exists the value e_crit, upon which there can be observed essential changes of the physiological and biochemical properties of the cells. The effect of medium flows upon the cells is characterized by index F_stress, which takes into account both, the possible presence of the cells in the zone with e ≧ e_crit, and the degree of medium flow and cell interaction, numerically equal to value (e – e_crit)^0.5.     

Clustering of mutations affecting alginic acid biosynthesis in mucoid Pseudomonas aeruginosa.

A 10-kilobase DNA fragment previously shown to contain the phosphomannose isomerase gene (pmi) of Pseudomonas aeruginosa was used to construct a pBR325-based hybrid that can be propagated in P. aeruginosa only by the formation of a chromosomal-plasmid cointegrate. This plasmid, designated pAD4008, was inserted into the P. aeruginosa chromosome by recombination at a site of homology between the cloned P. aeruginosa DNA and the chromosome. Mobilization of pAD4008 into P. aeruginosa PAO and 8830 and selection for the stable acquisition of tetracycline resistance resulted in specific and predictable changes in the pattern of endonuclease restriction sites in the phosphomannose isomerase gene region of the chromosomes. Chromosomal DNA from the tetracycline-resistant transformants was used to clone the drug resistance determinant with Bg/II or XbaI, thereby allowing the "walking" of the P. aeruginosa chromosome in the vicinity of the pmi gene. Analysis of overlapping tetracycline-resistant clones indicated the presence of sequences homologous to the DNA insert of plasmid pAD2, a recombinant clone of P. aeruginosa origin previously shown to complement several alginate-negative mutants. Restriction mapping, subcloning, and complementation analysis of a 30-kilobase DNA region demonstrated the tight clustering of several genetic loci involved in alginate biosynthesis. Furthermore, the tetracycline resistance determinant in PAO strain transformed by pAD4008 was mapped on the chromosome by plasmid FP2-mediated conjugation and was found to be located near 45 min.     

Plasmodium subtilisin-like protease 1 (SUB1): insights into the active-site structure, specificity and function of a pan-malaria drug target

Release of the malaria merozoite from its host erythrocyte (egress) and invasion of a fresh cell are crucial steps in the life cycle of the malaria pathogen. Subtilisin-like protease 1 (SUB1) is a parasite serine protease implicated in both processes. In the most dangerous human malarial species, Plasmodium falciparum, SUB1 has previously been shown to have several parasite-derived substrates, proteolytic cleavage of which is important both for egress and maturation of the merozoite surface to enable invasion. Here we have used molecular modelling, existing knowledge of SUB1 substrates, and recombinant expression and characterisation of additional Plasmodium SUB1 orthologues, to examine the active site architecture and substrate specificity of P. falciparum SUB1 and its orthologues from the two other major human malaria pathogens Plasmodium vivax and Plasmodium knowlesi, as well as from the rodent malaria species, Plasmodium berghei. Our results reveal a number of unusual features of the SUB1 substrate binding cleft, including a requirement to interact with both prime and non-prime side residues of the substrate recognition motif. Cleavage of conserved parasite substrates is mediated by SUB1 in all parasite species examined, and the importance of this is supported by evidence for species-specific co-evolution of protease and substrates. Two peptidyl alpha-ketoamides based on an authentic PfSUB1 substrate inhibit all SUB1 orthologues examined, with inhibitory potency enhanced by the presence of a carboxyl moiety designed to introduce prime side interactions with the protease. Our findings demonstrate that it should be possible to develop 'pan-reactive' drug-like compounds that inhibit SUB1 in all three major human malaria pathogens, enabling production of broad-spectrum antimalarial drugs targeting SUB1.     

Studies of the mixing character and flow distribution in mycelial fermentation broths

The influence of two mixing systems on the principal parameters of mycelial fermentations of Aspergillus niger, Fusicoccum amygdali Del. and Fusarium moniliforme Sheld. as well as their metabolite citric acid, fusicoccin and gibberellic acid production was analyzed from the viewpoint of flow energy distribution in a bioreactor. The growth and metabolite synthesis during fermentation was compared under different mixing conditions in the fermenter FU-8 with a turbine mixing system (TMS) and a counterflow mixing system (CMS). It was found that the growth, productivity and respiration characteristics as well as the morphology of these cultures varied dependent on the mixing system and agitation regime used. The counterflow mixing system was more favourable for large agglomerates (F. amygdali) or soft pellets (A. niger) forming fungi, while the turbine mixing system was more effective for F. moniliforme growing in the form of small clumps and freely dispersed hyphae. Flow characteristics under different mixing conditions were analyzed in a model fermenter. The kinetic energy of flow fluctuations was measured in gassed and ungassed water and different fermentation broth systems by using a Stirring Intensity Measuring Device (SIMD-F1). The difference of the energy values at different points was better expressed in the fermenter with a turbine mixing system in comparison with that having a counterflow mixing system. High viscous F. amygdali and A. niger broth provided higher energy values compared to water and low viscous F. moniliforme broth. It was observed that the intensity of growth and the intensity of the synthesis decreased at very high energy values, which was obviously connected to the influence of the irreversible shear stress on the mycelial morphology.     

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.     

Biomechanical properties of oesophagus wall under loading

In this investigation, firstly, the biomechanical properties of different parts of oesophagus were determined. Oesophagus stress and strain are the greatest in the cervical part for all age groups. The human oesophagus deforms unevenly, depending on the direction of load in relation to the organ's axis, it exhibits anisotropical behaviour. With the age the values of mechanical parameters of the oesophagus wall reduce, in particular beginning from 45 years of age, but the modulus of elasticity increases. Biomechanical properties of the oesophagus depend on the architecture of its structure. By loading the organ in the circumferential direction, microfibrilae rupture and deformation of the muscular fibres occurs. With increase of load, collagenous fibres straighten and microruptures in collagenous fibrilae occur. With stretching of oesophagus longitudinally, collagenous fibres partially preserve their wavy and helical configuration. Therefore, higher resistance of the oesophageal wall occurs in the longitudinal direction.     

Model‐based optimization and pO2 control of fed‐batch Escherichia coli and Saccharomyces cerevisiae cultivation processes

A model‐based approach for optimization and cascade control of dissolved oxygen partial pressure (pO₂) and maximization of biomass in fed‐batch cultivations is presented. The procedure is based on the off‐line model‐based optimization of the optimal feeding rate profiles and the subsequent automatic pO₂ control using a proposed cascade control technique. During the model‐based optimization of the process, feeding rate profiles are optimized with respect to the imposed technological constraints (initial and maximal cultivation volume, cultivation time, feeding rate range, maximal oxygen transfer rate and pO₂ level). The cascade pO₂ control is implemented using activation of cascades for agitation, oxygen enrichment, and correction of the preoptimized feeding rate profiles. The proposed approach is investigated in two typical fed‐batch processes with Escherichia coli and Saccharomyces cerevisiae. The obtained results show that it was possible to achieve sufficiently high biomass levels with respect to the given technological constraints and to improve controllability of the investigated processes.     

Ultrasensitivity by Molecular Titration in Spatially Propagating Enzymatic Reactions

Delineating design principles of biological systems by reconstitution of purified components offers a platform to gauge the influence of critical physicochemical parameters on minimal biological systems of reduced complexity. Here we unravel the effect of strong reversible inhibitors on the spatiotemporal propagation of enzymatic reactions in a confined environment in vitro. We use micropatterned, enzyme-laden agarose gels which are stamped on polyacrylamide films containing immobilized substrates and reversible inhibitors. Quantitative fluorescence imaging combined with detailed numerical simulations of the reaction-diffusion process reveal that a shallow gradient of enzyme is converted into a steep product gradient by addition of strong inhibitors, consistent with a mathematical model of molecular titration. The results confirm that ultrasensitive and threshold effects at the molecular level can convert a graded input signal to a steep spatial response at macroscopic length scales.     

Decreased Silica Land–sea Fluxes through Damming in the Baltic Sea Catchment – Significance of Particle Trapping and Hydrological Alterations

We tested the hypothesis that reservoirs with low water residence time and autochthonous production influence river biogeochemistry in eutrophied river systems draining cultivated watersheds. The effect of a single artificial water reservoir and consecutive reservoirs on silica (Si) river fluxes is exemplified by the moderately dammed Vistula River and the heavily regulated Daugava River that are compared with the practically undammed Oder River. The sum of the discharge weighted annual mean biogenic silica (BSi) and dissolved silicate (DSi) concentrations in the rivers Oder, Vistula and Daugava were about 160 μ M (40 + 120 μ M), 150 μ M (20 + 130 μ M) and 88 μ M (6 + 82 μ M), respectively. Assuming BSi and DSi concentrations as observed in the Oder River as typical for eutrophied but undammed rivers, complete trapping of this BSi could have lowered Si fluxes to the Baltic Sea from rivers with cultivated watersheds by 25%. The superimposed effect of hydrological alterations on reduced Si land–sea fluxes is demonstrated by studies in the boreal/subarctic and oligotrophic rivers Kalixälven and Lueälven. The DSi yield of the heavily dammed Luleälven (793 kg km^?2 yr^?1) constituted only 63% of that was found in the unregulated Kalixälven (1261 kg km^?2 yr^?1), despite the specific runoff of the Luleälven (672 mm m^?2 yr^?1) being 19% higher than that of theKalixälven (563 mm m^?2 yr^?1); runoff normalized DSi yield of the former, regulated watershed, was only half the DSi yield of the latter, unperturbed watershed. Based on these findings, it is hypothesized here that perturbed surface water–groundwater interactions are the major reasons for the reduced annual fluctuations in DSi concentrations as also seen in the heavily dammed and eutrophic river systems such as the Daugava and Danube.