Rates of net nitrogen mineralization in disturbed and undisturbed soils

Quantification of net nitrogen mineralization (NNM) in soils is indispensable in order to optimize N fertilization of crops. Two long-term laboratory incubation methods were applied to determine rates of net nitrogen mineralization (r_NNM) of soils from two sites of arable land (sandy loam soil, silty loam soil) at four temperature levels (2°C, 8°C, 14°C, 21°C). Since variability within replicates was small, the modified 12-week incubation method of Stanford and Smith (1972) using disturbed soils allowed to establish reliable Arrhenius functions with reasonable expenditure. The fit of the functions derived from the 5-month incubation of 23 undisturbed soil columns (4420 cm³) was worse. This was caused by greater variability and less differentiation between temperature levels. Results of both experiments could be described best by zero-order kinetics. Mean mineralization rates of disturbed samples were approximately twice as high than those of undisturbed samples. The suitability of both methods for the prediction of NNM at site conditions is discussed. Actual respiration (AR) at incubation temperatures and substrate induced respiration (SIR) were measured at the end of the incubation of undisturbed soil columns. The results presented reveal that soil microbial communities develop in a different manner during long-term incubation at different temperatures. This behavior offends the underlying assumption that soil microbes remain in steady-state during incubation and that rising rates are physiological reactions to temperature enhancement. Therefore soil microbial biomass (SMB) dynamics during the experiment has to be accounted for when rates of NNM and Arrhenius functions are established. R Merck Section editor     

Preparation and characterization of flurbiprofen beads by melt solidification technique

Amelt solidification technique has been developed to obtain sustained-release waxy beads of flurbiprofen. Low glass transition temperature (t _g) and shear-induced crystallization of flurbiprofen made it a suitable candidate for melt solidification technique. The process involved emulsification and solidification of flurbiprofen-cetyl alcohol melt at significantly low temperature (5°C). The effect of variables, namely, the amount of cetyl alcohol and the speed of agitation, was studied using 3² factorial design. The technique and the beads were evaluated on the basis of process and desired yield, surface topography, Fourier-transform infrared (FT-IR), differential scanning calorimetry (DSC), particle size distribution, crushing strength, and drug release. Average values for process and desired yields were 97% wt/wt and 26% wt/wt, respectively. No interaction was observed between drug and excipient. Multiple regression analysis was carried out, and response surfaces were obtained. A curvilinear relationship was observed between percentage of desired yield and the amount of cetyl alcohol. Linear decrease in crushing strength was observed with increase in the amount of cetyl alcohol. Drug released from the beads followed zero order kinetics. Burst release was shown to a greater extent in beads containing a lower amount of cetyl alcohol. Response surfaces of time required for certain percentage of drug (t _D%) showed that after critical concentration of about 20% of cetyl alcohol (400 mg/batch), no significant release retardant effect was observed.     

Fast sampling,rapid filtration apparatus: Principal characteristics and validation from studies ofd-glucose transport in human jejunal brush-border membrane vesicles

Summary Kinetic data in (brush-border) membrane vesicles which rely on the validity of the initial rate assumption for their interpretation and depend on tracer flux studies using the rapid filtration technique for their experimental measurement have been limited to some extent by the absence of techniques that would allow for real-time data analysis. In this paper, we report on our successful design of a fast sampling, rapid filtration apparatus (FSRFA) which seems to fill up this technical gap since showing the following characteristics: (i) rapid injection (5 msec) and mixing (less than 100 msec) of small amounts of vesicles (10–40 l) with an incubation medium (0.2–1.0 ml); (ii) fast (20 to 80 msec depending on the sample volume) and multiple (up to 18 samples at a maximal rate of 4/sec) sampling of the uptake mixture followed by rapid quenching in the stop solution (approximately 5 msec) according to a predetermined time schedule (any time combination from 0.25 to 9999 sec); and (iii) fast, automated, and sampling-synchronized filtration and washings of the quenched uptake medium (only 15–20 sec are necessary for the first filtration followed by two washings and extra filtrations). As demonstrated using adult human jejunal brush-border membrane vesicles and Na⁺-d-glucose cotransport as models, the FSRFA accurately reproduces the manual aspects of the rapid filtration technique while allowing for very precise initial rate determinations. Moreover, the FSRFA has also been designed to provide as much versatility as possible and, in its present version, allows for a very precise control of the incubation temperature and also permits a few efflux protocols to be performed. Finally, its modular design, which separates the fast sampling unit from the rapid filtration device, should help in extending its use to fields other than transport measurement.     

A Study on the Synthetic Characteristics of the Extracellular Polysaccharide (EPS) of Ganoderma lucidum Cultured in Batch Fermentation Using a Kinetic Model

The synthetic characteristics of the extracellular polysaccharide (EPS) of Ganoderma lucidum in batch fermentation were studied. The result showed that the production of EPS was partially growth-associated. The cell dry weight (CDW) and EPS reached 15.56 g·L⁻¹ and 3.02 g·L⁻¹, respectively. The yield of EPS to cell dry weight (Y_p/x) was 0.19. On the basis of the test results of batch fermentation, a kinetic model was proposed by using the Logistic equation for cell growth, the Luedeking–Piret equation for EPS production, and the Luedeking-piret-like equation for the consumption of glucose as substrate. The calculated results using these models were satisfactorily compared with the experimental data under various concentrations of glucose, and the average of relative errors was found to be not more than 5%. The kinetic model had practical guidance interesting in producing PES by Ganoderma lucidum.     

Spectrophotometric determination of peptide transport with chromogenic peptide mimetics

A spectrophotometric assay to determine peptide transport has been developed. Using two chromogenic peptide mimetics, L-phenylalanyl-L-2-sulfanilylglycine (PSG) and L-phenylalanyl-L-3-thiaphenylalanine (PSP), the peptide transport patterns in individual cell species can be evaluated effectively. After the addition of PSG to a HeLa cell suspension, sulfanilic acid accumulated progressively inside, but not outside, the cells, demonstrating that PSG was transported wholly intact. The addition of PSP to the same cell suspension was followed immediately by extracellular thiophenol production. Measurement of the rate of thiophenol release thereby provided direct determination of PSP transport. The thiophenol release was consistent with Michaelis-Menten kinetics, with a K(m) of 0.016 mM and a V(max) of 5.07 nmol/min (1 x 10(6) cells/ml, pH 7.4, 37 degrees C). The resulting kinetic constants estimated were in agreement with values determined by single-substrate enzyme kinetics. Using PSP, transport kinetics of various dipeptides was examined by competitive spectrophotometry. As a result, dipeptides tested could be ranked in order of kinetic power for their transport.     

Reversibility and binding kinetics of Thermobifida fusca cellulases studied through fluorescence recovery after photobleaching microscopy

Cellulases are enzymes capable of depolymerizing cellulose. Understanding their interactions with cellulose can improve biomass saccharification and enzyme recycling in biofuel production. This paper presents a study on binding and binding reversibility of Thermobifida fusca cellulases Cel5A, Cel6B, and Cel9A bound onto Bacterial Microcrystalline Cellulose. Cellulase binding was assessed through fluorescence recovery after photobleaching (FRAP) at 23, 34, and 45 °C. It was found that cellulase binding is only partially reversible. For processive cellulases Cel6B and Cel9A, an increase in temperature resulted in a decrease of the fraction of cellulases reversibly bound, while for endocellulase Cel5A this fraction remained constant. Kinetic parameters were obtained by fitting the FRAP curves to a binding-dominated model. The unbinding rate constants obtained for all temperatures were highest for Cel5A and lowest for Cel9A. The results presented demonstrate the usefulness of FRAP to access the fast binding kinetics characteristic of cellulases operating at their optimal temperature.     

Isoforms of Na,K-ATPase inArtemia salina: II. Tissue distribution and kinetic characterization

Summary To characterize the molecular properties conveyed by the isoforms of the subunit of Na,K-ATPase, the two major transepithelial transporting organs in the brine shrimp (Artemia salina), the salt glands and intestines, were isolated in pure form. The isoforms were quantified by ATP-sensitive fluorescein isothiocyanate (FITC) labeling. The salt gland enzyme exhibits only the 1 isoform, whereas the intestinal enzyme exhibits both the 1 and the 2 isoforms. After 32 hours of development, Na,K-ATPase activity [in mol P_i/mg protein/hr (1u)] in whole homogenates was 32±6 in the salt glands and 12±3 in the intestinal preparations (mean±sem). The apparent half-maximal activation constants (K _1/2) of the salt gland enzyme as compared to the intestinal enzyme were 3.7±0.6mm vs. 23.5±4mm (P<0.01) for Na⁺, 16.6±2.2mm vs. 8.29±1.5mm for K⁺ (P<0.01), and 0.87±0.8mm vs. 0.79±1.1mm for ATP (NS). The apparentK _i's for ouabain inhibition were 1.1×10^–4 m vs. 2×10^–5 m, respectively. Treatment of whole homogenates with deoxycholic acid (DOC) produced a maximal Na,K-ATPase activation of 46% in the salt gland as compared to 23% in the intestinal enzyme. Similar differences were found with sodium dodecyl sulfate (SDS). The two distinct forms of Na,K-ATPase isolated from the brine shrimp differed markedly in three kinetic parameters as well as in detergent sensitivity. The differences inK _1/2 for Na⁺ and K⁺ are more marked than those reported for the mammalian Na,K-ATPase isoforms. These differences may be attributed to the relative abundances of the subunit isoforms; other potential determinants (e.g. differences in membrane lipids), however, have not been investigated.During the tenure of an Educational Commission For Foreign Medical Graduates Visiting Associate Professorship.     

Mn2+ reduces Yz + in manganese-depleted Photosystem II preparations

Manganese in the oxygen-evolving complex is a physiological electron donor to Photosystem II. PS II depleted of manganese may oxidize exogenous reductants including benzidine and Mn²⁺. Using flash photolysis with electron spin resonance detection, we examined the room-temperature reaction kinetics of these reductants with Y_z ⁺, the tyrosine radical formed in PS II membranes under illumination. Kinetics were measured with membranes that did or did not contain the 33 kDa extrinsic polypeptide of PS II, whose presence had no effect on the reaction kinetics with either reductant. The rate of Y_z ⁺ reduction by benzidine was a linear function of benzidine concentration. The rate of Y_z ⁺ reduction by Mn²⁺ at pH 6 increased linearly at low Mn²⁺ concentrations and reached a maximum at the Mn²⁺ concentrations equal to several times the reaction center concentration. The rate was inhibited by K⁺, Ca²⁺ and Mg²⁺. These data are described by a model in which negative charge on the membrane causes a local increase in the cation concentration. The rate of Y_z ⁺ reduction at pH 7.5 was biphasic with a fast 400 s phase that suggests binding of Mn²⁺ near Y_z ⁺ at a site that may be one of the native manganese binding sites.Abbreviations PS II Photosystem II - Y_D tyrosine residue in Photosystem II that gives rise to the stable Signal II EPR spectrum - Y_z tyrosine residue in Photosystem II that mediates electron transfer between the reaction center chlorophyll and the site of water oxidation - ESR electron spin resonance - DPC diphenylcarbazide - DCIP dichlorophenolindophenol     

The major outer membrane protein of Fusobacterium nucleatum (FomA) folds and inserts into lipid bilayers via parallel folding pathways

Membrane protein insertion and folding was studied for the major outer membrane protein of Fusobacterium nucleatum (FomA), which is a voltage-dependent general diffusion porin. The transmembrane domain of FomA forms a beta-barrel that is predicted to consist of 14 beta-strands. Here, unfolded FomA is shown to insert and fold spontaneously and quantitatively into phospholipid bilayers upon dilution of the denaturant urea, which was shown previously only for outer membrane protein A (OmpA) of Escherichia coli. Folding of FomA is demonstrated by circular dichroism and fluorescence spectroscopy, by SDS-polyacrylamide gel electrophoresis, and by single-channel recordings. Refolded FomA had a single-channel conductance of 1.1 nS at 1 M KCl, in agreement with the conductance of FomA isolated from membranes in native form. In contrast to OmpA, which forms a smaller eight-stranded beta-barrel domain, folding kinetics of the larger FomA were slower and provided evidence for parallel folding pathways of FomA into lipid bilayers. Two pathways were observed independent of membrane thickness with two different lipid bilayers, which were either composed of dicapryl phosphatidylcholine or dioleoyl phosphatidylcholine. This is the first observation of parallel membrane insertion and folding pathways of a beta-barrel membrane protein from an unfolded state in urea into lipid bilayers. The kinetics of both folding pathways depended on the chain length of the lipid and on temperature with estimated activation energies of 19 kJ/mol (dicapryl phosphatidylcholine) and 70 kJ/mol (dioleoyl phosphatidylcholine) for the faster pathways.