Computer Simulation of Assembly and Co-operativity of Hexameric AAA ATPases

AAA ATPases form a functionally diverse superfamily of proteins. Most members form homo-hexameric ring complexes, are catalytically active only in the fully assembled state, and show co-operativity among the six subunits. The mutual dependence among the subunits is clearly evidenced by the fact that incorporation of mutated, inactive subunits can decrease the activity of the remaining wild type subunits. For the first time, we develop here models to describe this form of allostery, evaluate them in a simulation study, and test them on experimental data. We show that it is important to consider the assembly reactions in the kinetic model, and to define a formal inhibition scheme. We simulate three inhibition scenarios explicitly, and demonstrate that they result in differing outcomes. Finally, we deduce fitting formulas, and test them on real and simulated data. A non-competitive inhibition formula fitted experimental and simulated data best. To our knowledge, our study is the first one that derives and tests formal allosteric schemes to explain the inhibitory effects of mutant subunits on oligomeric enzymes.     

Loci controlling lymphocyte production of interferon γ after alloantigen stimulation in vitro and their co-localization with genes controlling lymphocyte infiltration of tumors and tumor susceptibility

Low infiltration of lymphocytes into cancers is associated with poor prognosis, but the reasons why some patients exhibit a low and others a high infiltration of tumors are unknown. Previously we mapped four loci (Lynf1–Lynf4) controlling lymphocyte infiltration of mouse lung tumors. These loci do not encode any of the molecules that are involved in traffic of lymphocytes. Here we report a genetic relationship between these loci and the control of production of IFNγ in allogeneic mixed lymphocyte cultures (MLC). We found that IFNγ production by lymphocytes of O20/A mice is lower than by lymphocytes of OcB-9/Dem mice (both H2 ^pz ) stimulated in MLC by irradiated splenocytes of C57BL/10SnPh (H2 ^b ) or BALB/cHeA (H2 ^d ) mice, or by ConA. IFNγ production in MLCs of individual (O20 × OcB-9)F₂ mice stimulated by irradiated C57BL/10 splenocytes and genotyped for microsatellite markers revealed four IFNγ-controlling loci (Cypr4-Cypr7), each of which is closely linked with one of the four Lynf loci and with a cluster of susceptibility genes for different tumors. This suggests that inherited differences in certain lymphocyte responses may modify their propensity to infiltrate tumors and their capacity to affect tumor growth.     

Vascular supply of the anterior interventricular epicardial nerves and ventricular Purkinje fibers in the porcine hearts

The aim of this study was to perform a pilot histological and quantitative analysis of the blood vessels accompanying the epicardial nerves (vasa nervorum) in the porcine hearts. Twenty healthy porcine hearts were used in this study. The blood vessels were analyzed by light microscopy using four different staining techniques in transverse sections taken from the upper, middle, and lower segments of the anterior part of the interventricular region and the adjacent parts of the right and left ventricles containing epicardial nerves and the endocardial peripheral parts of the Purkinje fibers. In total, 317 epicardial nerves were detected. The vasa nervorum were present in 75.7% of these nerves. The vasa nervorum resembled arterioles and postcapillary and collecting venules. One hundred and forty nine epicardial nerves were perivascular, located in the adventitia of the anterior interventricular artery and vein. The remaining 168 nerves ran freely through the epicardial interstitium. The presence of the vasa nervorum was not related to topographical location or nerve diameter. Additionally, from a total of 33 analyzed ventricular complexes of Purkinje fibers small blood vessels located in their proximity were identified in only two cases. It can be concluded that the majority of the anterior epicardial nerves of porcine heart possess well-developed vasa nervorum. In contrast, similar blood vessels are rarely present in the vicinity of the Purkinje fibers. The data obtained contribute to a better understanding of the nutrition of the cardiac nerves.     

Electrochemical identification of microbially mediated hydrogen sulfide oxidation

The process of H₂S oxidation by the phototrophic bacteriaThiocapsa roseopersicina andChlorobium phaeobacteroides, respectively, was monitored using a Pt-glass-Ag⁰, Ag₂S electrode combination without liquid junction. Due to the resulting pe(pH) and pH₂S plottings three steps can be distinguished: oxidation of H₂S to an S(0) state, oxidation of S (0) to SO₄ ^2–, and oxidation of the remaining H₂S directly to SO₄ ^2–. Differences between the investigated bacteria exist with respect to their individual oxidation strategies.Thiocapsa apparently stops oxidizing H₂S at pH₂S 7.5 (e.g. 10^–7.5M H₂S) and shifts to the utilization of the intracellularly stored S (0). In contrastChlorobium utilizes its extracellularly stored sulfur parallel to the extracellular H₂S fraction. The corresponding Pt-sensor responses (pe₇ values) were found to be similar to the corresponding partial redox equilibria (p₇ values) of H₂S oxidation stoichiometries as proposed by Van Niel (1931) and Trüper (1964). It is concluded that the recording of pe enables investigators to understand (and control) in situ redox processes, independent of their thermodynamic equilibration, only bound to changes of electroactivity vs. sensor.     

Continuous acetone-butanol production with direct product removal

Summary To eliminate the product inhibition and increase the productivity of butanol formation, a continuously operated membrane bioreactor was connected to a four-stage mixer-settler cascade. Clostridium acetobutylicum was cultivated in this reactor. Butanol was selectively extracted with butyric acid saturated n-decanol from the cell-free cultivation medium, and the butanol-free medium was refed into the reactor. Due to the high boiling point of decanol, the recovery of butanol from the decanol solution is easy. The partition coefficient and selectivity of butanol in the cultivation medium-decanol-system is sufficiently high for removing it from the medium. Direct contact of the cells with the decanol phase causes cell damage. However, decanol is practically insoluble in the fermentation medium, thus the contact of the cell-free medium with the solvent phase does not influence of cell growth and product formation. At a dilution rate of D _z=0.1 h^-1, the butanol productivity was increased by removing butanol from the medium by a factor of four. A further increase was prevented by a contaminant of the technical decanol, which was identified by GC-MS-analysis as 1-,3-hexandiol.Symbols D dilution rate, h^-1 - D _eff effective dilution rate (Eq. 3), h^-1 - D _Ex extraction dilution rate (Eq. 3), h^-1 - D _g dilution rate of cell suspension in reactor-filter-system, h^-1 - E degree of extraction (Eq. 3), l - P product concentration in medium after extraction, g l^-1 - P _O product concentration in reactor, g l^-1 - R _P productivity and product formation rate, g l^-1 h^-1 - q _p S specific product formation coefficient with regard to the cell growth rate, l - V _F volume of cell suspension in filter module, l - V _g volume of the cell suspension in reactor and in filter module V _g =V _R +V _F , l - V _R volume of cell suspension in ractor, l - v _O cell free feed rate, l h^-1 - v ₁ flow rate of cell suspension leaves the reactor, l h^-1 - v _E flow rate of decanol through the extractor, l h^-1 - v _w flow rate of the cell free medium through the filter modul, l h^-1 - X cell mass concentration, g l^-1 - specific growth rate of the cells, h^-1 Dedicated to Professor Dr. H. J. Rehm on the occasion of his 60th birthday     

Contribution of leaves of different ages to plant carbon balance as affected by potassium supply and water stress

The carbon balances of whole, 21-d old French bean plants (Phaseolus vulgaris L.) grown in standard nutrient solution (1K) and its modifications without (OK) or surplus (2K) potassium were calculated from the daily photosynthetic carbon inputs of individual leaves, and the daily respiratory carbon losses by individual leaves, stalks and petioles, and roots. Under the three K concentrations, maximum net photosynthetic rates (P_n) were found in the 2nd or in the 3rd trifoliate leaves, maximum respiratory rates (R_d) in the youngest, 4th trifoliate leaves; the P_n/R_d ratio decreased with leaf age. In all leaves of 2K plants, leaf dry masses and thicknesses, P_n, P_n/P_d ratios, and stomatal and intracellular conductances were lower than in OK and IK plants. Daily whole-plant net carbon gain was highest in IK plants, whereas in OK and 2K plants it was 98.0 and 81.3 % of IK, respectively. Similar values were found in the parameters of growth analysis, namely in net assimilation rates and relative growth rates. No differences were found in water potential (Ψ _w ) or water saturation deficit (W_sat) in the OK, 1K and 2K plants sufficiently supplied with water or during wilting and resaturation. The decrease in Ψ_w to −0.97 MPa was associated with a 19.9 %, 31.4 % and 23.4 % decrease in P_n of OK, 1K and 2K plants, respectively, but no effect on R_d was found. In the three variants, the short-time effect of mild water stress was fully reversible.