Avoidance of oxidative-stress perturbation in yeast bioprocesses by proteomic and genomic biostrategies?

AIMS: Bioprocess oxidative stress caused by many reactive oxygen species (ROS) can lead to largely irreversible perturbation of yeast bioprocesses. These include the production of proteins derived from recombinant DNA yeast technology (aerobically grown Saccharomyces cerevisiae). These proteins include rennin, amyloglucosidases (glucamylases), interferons, interleukins, insulin, monoclonal antibodies, tissue plasminogen activators (t-PA), sexually transmitted disease antigens, and measles, mumps and rubella antigens, growth hormones, somatotropin, blood clotting factors VIII and XIII. In addition, there may be a demand for severe acute respiratory syndrome-coronavirus antigens, hepatitis A, B and C viral-selected antigens, HIV retroviral antigens, influenza antigens, trypanosomal antigens, and foot and mouth disease antigens. Prevention of oxidative stress has been achieved by application of antioxidant redox metalloenzymes such as superoxide dismutases (containing Cu/Zn cytosolic, Mn mitochondrial and Fe bacterial) glutathione peroxidases (and other Se-containing proteins and enzymes such as the thioredoxins), catalases (Fe-containing), cytochrome c peroxidases (Fe-containing), ceruloplasmins (Cu-containing), metallothionines (these cysteine thiol-rich proteins bind ions of cadmium and mercury) and tyrosinases(Cu-containing). METHODS AND RESULTS: ROS are generated inadvertently by single metal valency couples such as FeII/FeIII and by FeIII/FeV present in 2700 (including 57 human) isoforms in cytochromes P450 mixed-function oxidases (EC 1.14.14.1; O2 : mono-oxygenase NADPH/NADH requiring). In addition, mixed-metal couples such as valency unmatched forms in CuI/FeII and FeIII/MnIV can recycle electrons. Moreover, proteins/protein chaperone couples can recycle electrons, often where futile-recycling systems have been instigated. Furthermore, oxidized membrane phospholipids (R) can form ROOH (lipid hydroperoxides) and ROH (lipid alkoxides) that can generate ROS through Fenton chemistry (iron-catalysed) chain reactions. Utilization of chain-breaking antioxidants such as vitamin E (alpha-tocopherol) in the lipid phase and vitamin C (ascorbate) in the aqueous phase can terminate these ROS-producing reactions. CONCLUSIONS: The main significance of the study is that proteomic strategies of relief from bioprocess perturbation by ROS of yeast fermentations (used to manufacture proteins required in the food and therapeutic bioindustries) may become possible through addition of selected proteins (including metalloenzymes). The main impact of the study is that the utilization of genetically modified (GM) yeast produced by recombinant DNA technology genomic strategies could circumvent the bioprocessing problems that otherwise result from the bioprocess perturbations: this is as a result of oxidative stress caused by ROS, which is avoidable by deployment of appropriate antioxidants such as vitamins E, C and D (and antioxidant proteins and enzymes often of microbial origin via recombinant DNA technology).     

Kinetic stabilization of the native state by protein engineering: implications for inhibition of transthyretin amyloidogenesis

The amyloidogenic homotetrameric protein transthyretin (TTR) must undergo rate-limiting dissociation to partially denatured monomers in order to aggregate. TTR contains two distinct quaternary interfaces, one of which defines the binding sites for thyroxine and small-molecule amyloidogenesis inhibitors. Kinetic stabilization of the tetramer can be accomplished either by the binding of amyloidogenesis inhibitors selectively to the native state over the dissociative transition state or by the introduction of trans-suppressor subunits (T119M) into heterotetramers to destabilize the dissociative transition state. In each case, increasing the dissociation activation barrier prevents tetramer dissociation. Herein, we demonstrate that tethering two subunits whose quaternary interface defines the thyroxine binding site also dramatically increases the barrier for tetramer dissociation, apparently by destabilization of the dissociative transition state. The tethered construct (TTR-L-TTR)2 is structurally and functionally equivalent to wild-type TTR. Urea is unable to denature (TTR-L-TTR)2, yet it is able to maintain the denatured state once denaturation is achieved by GdnHCl treatment, suggesting that (TTR-L-TTR)2 is kinetically rather than thermodynamically stabilized, consistent with the identical wild-type TTR and (TTR-L-TTR)2 GdnHCl denaturation curves. Studies focused on a construct containing a single TTR-L-TTR chain and two normal monomer subunits establish that alteration of only one quaternary structural interface is sufficient to impose kinetic stabilization on the entire quaternary structure.     

Structural characterization of the Ser324Thr variant of the catalase-peroxidase (KatG) from Burkholderia pseudomallei

The Ser315Thr variant of the catalase-peroxidase KatG from Mycobacterium tuberculosis imparts resistance to the pro-drug isonicotinic acid hydrazide (isoniazid) through a failure to convert it to the active drug, isonicotinoyl-NAD. The equivalent variant in KatG from Burkholderia pseudomallei, Ser324Thr, has been constructed, revealing catalase and peroxidase activities that are similar to those of the native enzyme. The other activities of the variant protein, including the NADH oxidase, the isoniazid hydrazinolysis and isonicotinoyl-NAD synthase activities are reduced by 60-70%. The crystal structure of the variant differs from that of the native enzyme in having the methyl group of Thr324 situated in the entrance channel to the heme cavity, in a modified water matrix in the entrance channel and heme cavity, in lacking the putative perhydroxy modification on the heme, in the multiple locations of a few side-chains, and in the presence of an apparent perhydroxy modification on the indole nitrogen atom of the active-site Trp111. The position of the methyl group of Thr324 creates a constriction or narrowing of the channel leading to the heme cavity, providing an explanation for the lower reactivity towards isoniazid and the slower rate of isonicotinoyl-NAD synthesis.     

A rapid algorithm for processing digital physiologic signals: Application to skeletal muscle contractions

Quantifying mechanical output is fundamental to understanding metabolism that fuels muscle contraction and more recent attempts to understand signal transduction and gene regulation. The latter requires long-term application of exercise protocols that result in large amounts of data on muscle performance. The purpose of this study was to develop software for automated quantification of skeletal muscle contractions. An in situ mouse sciatic nerve stimulation model was used to produce contractions over a broad range of frequencies and recorded as both digital and analog signals using a PC analog to digital converter board and chart recorder, respectively. Spectral analysis of the noise components formed the basis for designing a smoothing Chebyshev filter. Algorithms implemented in custom software identified twitches and estimated baseline levels from the smoothed signal. The time to peak force, peak force, tension-time integral, and half-relaxation time were determined for each twitch after baseline correction. The automated results were compared to those obtained from manual measurements of the analog signal. Bland–Altman analysis of the parameters computed from digital signals compared with the corresponding measurements by manual planometry demonstrates the agreement of the digital processing algorithm with planometry over a wide range of twitch characteristics. This program may also be used to study the mechanics of other preparations from isolated muscles, human proximal limb performance, and other digital physiologic signals. Adaptation of the filter function is required to apply the analysis to another experimental apparatus with differing noise characteristics. A full version of the program and instructions for its use are available for download at www.rad.msu.edu.     

Sampling effects, noise, and photobleaching in temporal image correlation spectroscopy

We present an extensive investigation of the accuracy and precision of temporal image correlation spectroscopy (TICS). Using simulations of laser scanning microscopy image time series, we investigate the effect of spatiotemporal sampling, particle density, noise, sampling frequency, and photobleaching of fluorophores on the recovery of transport coefficients and number densities by TICS. We show that the recovery of transport coefficients is usually limited by spatial sampling, while the measurement of accurate number densities is restricted by background noise in an image series. We also demonstrate that photobleaching of the fluorophore causes a consistent overestimation of diffusion coefficients and flow rates, and a severe underestimation of number densities. We derive a bleaching correction equation that removes both of these biases when used to fit temporal autocorrelation functions, without increasing the number of fit parameters. Finally, we image the basal membrane of a CHO cell with EGFP/alpha-actinin, using two-photon microscopy, and analyze a subregion of this series using TICS and apply the bleaching correction. We show that the photobleaching correction can be determined simply by using the average image intensities from the time series, and we use the simulations to provide good estimates of the accuracy and precision of the number density and transport coefficients measured with TICS.     

Reliability of video taped interviews with children suspected of being sexually abused.

OBJECTIVE--To determine the reliability of judgments about the likelihood of child sexual abuse based only on video recorded interviews. DESIGN--Blinded rating of likelihood of abuse by seven professional groups and comparison with consensus rating. SETTING--Child and adolescent psychiatry centre. SUBJECTS--Four people from each of seven professional disciplines: specialist psychiatrists, general psychiatrists, experimental psychologists, trainee social workers, trainee clinical psychologists, lawyers, and police. MAIN OUTCOME MEASURE--Rating of 12 recorded interviews. RESULTS--Agreement between the consensus panel and professional groups was 83% (151/183) for high likelihood cases (seven cases) and 89% (118/132) for low likelihood cases (five). Specialist psychiatrists and the police were better able to identify high likelihood cases than were other groups with less experience of interviewing sexually abused children (91% (48/53) v 79% (102/129); p = 0.05). CONCLUSIONS--Raters could accurately distinguish children with low likelihood of abuse on interview evidence alone, but those with more experience of dealing with sexual abuse were better at identifying high likelihood cases.     

The specificity of two proteinases that cleave adjacent to arginine, C1 esterase and acrosin, for peptide p-nitroanilide substrates

Relative values of Vmax/Km for hydrolysis of 40 peptide p-nitroanilides catalyzed by human Cl-s and human acrosin are reported. For Cl-s, Ac-Lys(gamma Cbz)-Gly-Arg is the optimum sequence, but 25% of the substrates have (Vmax/Km)rel greater than 0.25 compared to this sequence. The best acrosin substrate tested has the sequence Tos-Gly-Pro-Arg, although (Vmax/Km)rel greater than 0.15 for more than half of the substrates. Proline at P2 is preferred by acrosin. Both enzymes prefer arginine at P1 greater than or equal to 3-fold over lysine and will not accept citrulline. In addition, occupancy of site S3 may yield an increase in Vmax/Km of greater than or equal to 10-fold with either enzyme, but many residues are accepted at S2, S3 and S4. Thus, an acrosin assay using Tos-Gly-Pro-Arg p-nitroanilide as a substrate is more than 20-times as sensitive as existing assays with blocked arginine derivatives.