Performance characteristics of a reversible immunosensor with a heterobifunctional enzyme conjugate as signal generator

Factors that control the performance of a reversible immunosensor with an analyte (progesterone)-enzyme (horseradish peroxidase) conjugate as signal generator have been investigated. The conjugate is used in conjunction with two antibodies, which are specific to progesterone and to horseradish peroxidase, immobilized on two spatially separated polypropylene mesh discs. The conjugate and two antibodies are confined to an internal compartment of a microdialyzer by a semipermeable membrane. The small analyte from an external medium permeates across the membrane into the internal compartment where the analyte concentration determines the relative amounts of the bound conjugate on the two solid surfaces. By measuring two signals from the conjugate bound at two separate sites, we experimentally obtained time-response curves to a concentration pulse of the external analyte. A mathematical (kinetic) model describing the sensor system was developed and used for the determination of rate-limiting factors. In semicontinuous monitoring of the analyte concentrations, operation of the immunosensor with the enzyme conjugate as signal generator required special attention to (a) enzyme stability, (b) analyte permeation (dependence on medium components), and (c) kinetics related to the different accessibility to the same antibody of the small analyte (to be measured) vs. the larger counterpart on the enzyme conjugate (for signal generation). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 221-231, 1997.     

Hold your breath beetle—Mites!

Respiratory gas exchange in insects occurs via a branching tracheal system. The entrances to the air‐filled tracheae are the spiracles, which are gate‐like structures in the exoskeleton. The open or closed state of spiracles defines the three possible gas exchange patterns of insects. In resting insects, spiracles may open and close over time in a repeatable fashion that results in a discontinuous gas exchange (DGE) pattern characterized by periods of zero organism‐to‐environment gas exchange. Several adaptive hypotheses have been proposed to explain why insects engage in DGE, but none have attracted overwhelming support. We provide support for a previously untested hypothesis that posits that DGE minimizes the risk of infestation of the tracheal system by mites and other agents. Here, we analyze the respiratory patterns of 15 species of ground beetle (Carabidae), of which more than 40% of individuals harbored external mites. Compared with mite‐free individuals, infested one's engaged significantly more often in DGE. Mite‐free individuals predominantly employed a cyclic or continuous gas exchange pattern, which did not include complete spiracle closure. Complete spiracle closure may prevent parasites from invading, clogging, or transferring pathogens to the tracheal system or from foraging on tissue not protected by thick chitinous layers.     

Microscale distribution of populations and activities of Nitrosospira and Nitrospira spp. along a macroscale gradient in a nitrifying bioreactor: quantification by in situ hybridization and the use of microsensors.

The change of activity and abundance of Nitrosospira and Nitrospira spp. along a bulk water gradient in a nitrifying fluidized bed reactor was analyzed by a combination of microsensor measurements and fluorescence in situ hybridization. Nitrifying bacteria were immobilized in bacterial aggregates that remained in fixed positions within the reactor column due to the flow regimen. Nitrification occurred in a narrow zone of 100 to 150 microm on the surface of these aggregates, the same layer that contained an extremely dense community of nitrifying bacteria. The central part of the aggregates was inactive, and significantly fewer nitrifiers were found there. Under conditions prevailing in the reactor, i.e., when ammonium was limiting, ammonium was completely oxidized to nitrate within the active layer of the aggregates, the rates decreasing with increasing reactor height. To analyze the nitrification potential, profiles were also recorded in aggregates subjected to a short-term incubation under elevated substrate concentrations. This led to a shift in activity from ammonium to nitrite oxidation along the reactor and correlated well with the distribution of the nitrifying population. Along the whole reactor, the numbers of ammonia-oxidizing bacteria decreased, while the numbers of nitrite-oxidizing bacteria increased. Finally, volumetric reaction rates were calculated from microprofiles and related to cell numbers of nitrifying bacteria in the active shell. Therefore, it was possible for the first time to estimate the cell-specific activity of Nitrosospira spp. and hitherto-uncultured Nitrospira-like bacteria in situ.     

Iminoribitol transition state analogue inhibitors of protozoan nucleoside hydrolases.

Nucleoside N-ribohydrolases from protozoan parasites are targets for inhibitor design in these purine-auxotrophic organisms. Purine-specific and purine/pyrimidine-nonspecific nucleoside hydrolases have been reported. Iminoribitols that are 1-substituted with meta- and para-derivatized phenyl groups [(1S)-substituted 1, 4-dideoxy-1,4-imino-D-ribitols] are powerful inhibitors for the nonspecific nucleoside N-ribohydrolases, but are weak inhibitiors for purine-specific isozymes [Parkin, D. W., Limberg, G., Tyler, P. C., Furneaux, R. H., Chen, X.-Y., and Schramm, V. L. (1997) Biochemisty 36, 3528-3534]. Binding of these inhibitors to nonspecific nucleoside hydrolase occurs primarily via interaction with the iminoribitol, a ribooxocarbenium ion analogue of the transition state. Weaker interactions arise from hydrophobic interactions between the phenyl group and the purine/pyrimidine site. In contrast, the purine-specific enzymes obtain equal catalytic potential from leaving group activation and ribooxocarbenium ion formation. Knowledge of the reaction mechanisms and transition states for these enzymes has guided the design of isozyme-specific transition state analogue inhibitors. New synthetic efforts have produced novel inhibitors that incorporate features of the leaving group hydrogen-bonding sites while retaining the iminoribitol group. These compounds provide the first transition state analogue inhibitors for purine-specific nucleoside hydrolase. The most inhibitory 1-substituted iminoribitol heterocycle is a sub-nanomolar inhibitor for the purine-specific nucleoside hydrolase from Trypanosoma brucei brucei. Novel nanomolar inhibitors are also described for the nonspecific nucleoside hydrolase from Crithidia fasciculata. The compounds reported here are the most powerful iminoribitol inhibitors yet described for the nucleoside hydrolases.     

Transition-state analogs as inhibitors of human and malarial hypoxanthine-guanine phosphoribosyltransferases.

The proposed transition state for hypoxanthine-guanine phosphoribosyltransferases (HGPRTs) has been used to design and synthesize powerful inhibitors that contain features of the transition state. The iminoribitols (1S)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol 5-phosphate (immucillinHP) and (1S)-1-(9-deazaguanin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol 5-phosphate (immucillinGP) are the most powerful inhibitors yet reported for both human and malarial HGPRTs. Equilibrium binding constants are >1,000-fold tighter than the binding of the nucleotide substrate. The NMR spectrum of malaria HGXPRT in the Michaelis complex reveals downfield hydrogen-bonded protons. The chemical shifts move farther downfield with bound inhibitor. The inhibitors are lead compounds for species-specific antibiotics against parasitic protozoa. The high-resolution crystal structure of human HGPRT with immucillinGP is reported in the companion paper.     

The 2.0 A structure of human hypoxanthine-guanine phosphoribosyltransferase in complex with a transition-state analog inhibitor.

The structure of human HGPRT bound to the transition-state analog immucillinGP and Mg2+-pyrophosphate has been determined to 2.0 A resolution. ImmucillinGP was designed as a stable analog with the stereoelectronic features of the transition state. Bound inhibitor at the catalytic site indicates that the oxocarbenium ion of the transition state is stabilized by neighboring-group participation from MgPPi and O5'. A short hydrogen bond forms between Asp 137 and the purine ring analog. Two Mg2+ ions sandwich the pyrophosphate and contact both hydroxyls of the ribosyl analog. The transition-state analog is shielded from bulk solvent by a catalytic loop that moves approximately 25 A to cover the active site and becomes an ordered antiparallel beta-sheet.     

Deciphering the physiological response of Escherichia coli under high ATP demand

One long‐standing question in microbiology is how microbes buffer perturbations in energy metabolism. In this study, we systematically analyzed the impact of different levels of ATP demand in Escherichia coli under various conditions (aerobic and anaerobic, with and without cell growth). One key finding is that, under all conditions tested, the glucose uptake increases with rising ATP demand, but only to a critical level beyond which it drops markedly, even below wild‐type levels. Focusing on anaerobic growth and using metabolomics and proteomics data in combination with a kinetic model, we show that this biphasic behavior is induced by the dual dependency of the phosphofructokinase on ATP (substrate) and ADP (allosteric activator). This mechanism buffers increased ATP demands by a higher glycolytic flux but, as shown herein, it collapses under very low ATP concentrations. Model analysis also revealed two major rate‐controlling steps in the glycolysis under high ATP demand, which could be confirmed experimentally. Our results provide new insights on fundamental mechanisms of bacterial energy metabolism and guide the rational engineering of highly productive cell factories.     

Analysis of Stomatal and Nonstomatal Components in the Environmental Control of CO(2) Exchange in Leaves of Welwitschia mirabilis

In well-watered plants of Welwitschia mirabilis, grown in the glass-house under high irradiance conditions, net CO₂ assimilation was almost exclusively observed during the daytime. The plants exhibited a very low potential for Crassulacean acid metabolism, which usually resulted in reduced rates of net CO₂ loss for several hours during the night. In leaves exposed to the diurnal changes in temperature and humidity typical of the natural habitats, CO₂ assimilation rates in the light were markedly depressed under conditions resembling those occurring during midday, when leaf temperatures and the leaf-air vapor pressure differences were high (36°C and 50 millibars bar⁻¹, respectively). Studies on the relationship between CO₂ assimilation rate and intercellular CO₂ partial pressure at various temperatures and humidities showed that this decrease in CO₂ assimilation was largely due to stomatal closure. The increase in the limitation of photosynthesis by CO₂ diffusion, which is associated with the strong decline in stomatal conductance in Welwitschia exposed to midday conditions, may significantly contribute to the higher ¹³C content of Welwitschia compared to the majority of C₃ species.