Tailoring the properties of (catalytically)-active inclusion bodies.

BACKGROUND: Immobilization is an appropriate tool to ease the handling and recycling of enzymes in biocatalytic processes and to increase their stability. Most of the established immobilization methods require case-to-case optimization, which is laborious and time-consuming. Often, (chromatographic) enzyme purification is required and stable immobilization usually includes additional cross-linking or adsorption steps. We have previously shown in a few case studies that the molecular biological fusion of an aggregation-inducing tag to a target protein induces the intracellular formation of protein aggregates, so called inclusion bodies (IBs), which to a certain degree retain their (catalytic) function. This enables the combination of protein production and immobilization in one step. Hence, those biologically-produced immobilizates were named catalytically-active inclusion bodies (CatIBs) or, in case of proteins without catalytic activity, functional IBs (FIBs). While this strategy has been proven successful, the efficiency, the potential for optimization and important CatIB/FIB properties like yield, activity and morphology have not been investigated systematically. RESULTS: We here evaluated a CatIB/FIB toolbox of different enzymes and proteins. Different optimization strategies, like linker deletion, C- versus N-terminal fusion and the fusion of alternative aggregation-inducing tags were evaluated. The obtained CatIBs/FIBs varied with respect to formation efficiency, yield, composition and residual activity, which could be correlated to differences in their morphology; as revealed by (electron) microscopy. Last but not least, we demonstrate that the CatIB/FIB formation efficiency appears to be correlated to the solvent-accessible hydrophobic surface area of the target protein, providing a structure-based rationale for our strategy and opening up the possibility to predict its efficiency for any given target protein. CONCLUSION: We here provide evidence for the general applicability, predictability and flexibility of the CatIB/FIB immobilization strategy, highlighting the application potential of CatIB-based enzyme immobilizates for synthetic chemistry, biocatalysis and industry.

Prehospital hydroxocobalamin for inhalation injury and cyanide toxicity in the United States - analysis of a database and survey of ems providers.

Prehospital use of hydroxocobalamin as an antidote for cyanide toxicity, a serious complication of smoke inhalation, has yet to be universally adopted in the United States though its efficacy and safety have been demonstrated since 2006. The purpose of this study was to characterize practices of prehospital hydroxocobalamin administration via a survey of emergency medical services (EMS) and to report a case series from an EMS database to track use of hydroxocobalamin. The Fire Smoke Coalition Newsletter emailed a voluntary survey to EMS subscribers regarding hydroxocobalamin use. Survey responses were analyzed in addition to survival data from the Smoke Inhalation Treatment Database (SITD), a publically available, self-reported, online database for EMS regarding smoke inhalation patient outcomes. Analysis was compared to current published data from PubMed. The survey had a 14% response rate (284/2000). Only 38% reported prehospital utilization of a hydrogen cyanide antidote with 46% using hydroxocobalamin. 20% of responders reported a formal ALS protocol was in place for hydroxocobalamin use. For the SITD, 12 of 13 (92%) patients who received hydroxocobalamin for suspected inhalation survived. Other studies found a survival rate of 72% and 42% after administration of hydroxocobalamin for smoke inhalation. Prehospital administration of hydroxocobalamin for cyanide toxicity is uncommon in the United States, as evidenced by this analysis, despite well-documented safety and efficacy. Although a small sample, patients who received prehospital hydroxocobalamin had improved survival. This survival rate is significantly greater than those reported previously.

L'utilisation préhospitalière de l'hydroxocobalamine (OHB12) comme antidote du cyanure, intoxication grave compliquant les inhalations de fumées (IF), n'est toujours pas réalisée partout aux États-Unis, bien que son innocuité et son efficacité aient été démontrées dès 2006. Les buts de cette étude était de caractériser l'utilisation préhospitalière d'OHB12 à travers les données des services d'urgences (SU) et de rapporter une série de cas issus de leurs dossiers. La « Fire Smoke Coalition Newsletter ¼ a proposé par courriel aux services d'urgence abonnés un étude sur l'utilisation d'OHB12, basée sur le volontariat. En plus des réponses, nous avons analysé les données de la « Smoke Inhalation Treatment Database ¼ (STID), banque de données publique abondée par les SU, colligeant le devenir des patients victimes d'une IF et les avons comparées aux données de la littérature, retrouvée dans PubMed. Le taux de réponse au questionnaire a été de 14% (284/2 000). Trente huit pour cent des répondeurs utilisent des antidotes au cyanure en préhospitalier, qui est OHB12 dans 46% des cas. Vingt pour cent des répondeurs attestent de l'existence d'un protocole formalisé quant à l'utilisation d'OHB12. Selon STID, 92% (12/13) des patients ayant reçu OHB12 ont survécu. D'autres études retrouvent des taux de survie de 72% et 42% après administration de OHB12 dans le cadre de l'IF. Cette étude confirme que l'utilisation préhospitalière d'OHB12 dans le cadre de l'intoxication au cyanure n'est pas habituelle aux États-Unis, malgré une efficacité et une innocuité reconnues. Bien que l'échantillon soit faible, les patients ayant reçu OHB12 en préhospitalier ont un taux de survie nettement amélioré par rapport à ceux précédemment rapportés.

HLA-A2.1/K(b) transgenic murine dendritic cells transduced with an adenovirus encoding human gp100 process the same A2.1-restricted peptide epitopes as human antigen-presenting cells and elicit A2.1-restricted peptide-specific CTL.

HLA-A2.1/K(b) transgenic mice (A2.1/K(b) mice) were used to investigate the processing of human gp100 melanoma antigen by murine antigen presenting cells (APC). Bone marrow-derived dendritic cells (DC) from A2.1/K(b) mice were transduced with adenovirus encoding human gp100 (Ad2/hugp100v2). The Ad2/hugp100v2-transduced DC express human gp100, as documented by immunoperoxidase staining. Flow cytometric analysis demonstrates that Ad vector transduction does not downregulate expression of several markers, including MHC class I. We show that Ad2/hugp100v2-transduced DC are recognized by peptide-specific, A2.1-restricted CTL, suggesting correct processing and presentation of the hugp100 antigen by murine DC. To assess dominance among the various A2.1-restricted epitopes encoded by hugp100, A2.1/K(b) transgenic mice were immunized with Ad2/hugp100v2-transduced DC. Resulting effector cytotoxic T lymphocytes (CTL) were assayed for peptide specificity using a panel of six synthetic peptides known to encode A2.1-restricted epitopes of human gp100 (denoted G154, G177, G209, G280, G457, G476). CTL obtained from Ad2/hugp100v2-transduced DC immunized A2.1/K(b) mouse lysed target cells presenting five of the six epitopes, supporting the observation that murine cells correctly process the hugp100 antigen. The immunogenicity of individual gp100 epitopes correlates with their binding affinity to A2.1. CTL generated from A2.1/K(b) mice immunized with Ad2/hugp100v2-transduced DC also specifically recognize A2.1(+)/gp100(+) human melanoma cells. These data suggest that murine APC process and present the same set of HLA-restricted peptides, similar to human APC. HLA transgenic mice serve as a useful model system to study class I-restricted epitopes of human tumor-associated antigens.

In vitro priming with adenovirus/gp100 antigen-transduced dendritic cells reveals the epitope specificity of HLA-A*0201-restricted CD8+ T cells in patients with melanoma.

Replication-deficient recombinant adenovirus (Ad) encoding human gp100 or MART-1 melanoma Ag was used to transduce human dendritic cells (DC) ex vivo as a model system for cancer vaccine therapy. A second generation E1/E4 region deleted Ad which harbors the CMV immediate-early promoter/enhancer and a unique E4-ORF6/pIX chimeric gene was employed as the backbone vector. We demonstrate that human monocyte-derived DC are permissive to Ad infection at multiplicity of infection between 100 and 500 and occurs independent of the coxsackie Ad receptor. Fluorescent-labeled Ad was used to assess the kinetics and distribution of viral vector within DC. Ad-transduced DC show peak transgene expression at 24-48 h and expression remains detectable for at least 7 days. DC transduced with replication-deficient Ad do not exhibit any unusual phenotypic characteristics or cytopathic effects. DC transduced with Ad2/gp100v2 can elicit tumor-specific CTL in vitro from patients bearing gp100+ metastatic melanoma. Using a panel of gp100-derived synthetic peptides, we show that Ad2/gp100v2-transduced DC elicit Ag-specific CTL that recognize only the G209 and G280 epitopes, both of which display relatively short half-lives ( approximately 7-8 h) on the surface of HLA-A*0201+ cells. Thus, patients with metastatic melanoma are not tolerant to gp100 Ag based on the detection of CD8+ T cells specific for multiple HLA-A*0201-restricted, gp100-derived epitopes.

Mismatch repair is diminished during stationary-phase mutation.

This paper is an invited Response to a recent Commentary [P.L. Foster, Rev. Mut. Res. 436 (1999) 179-184] entitled "Are adaptive mutations due to a decline in mismatch repair? The evidence is lacking". The Commentary argues that no evidence exists supporting the idea that mismatch repair is limiting specifically during stationary-phase mutation. A primary concern of the author is to question the method that we used previously to measure growth-dependent mutation. In this method, mutation rates are calculated using counts of mutant colonies taken at times when those colonies arise, rather than at a predetermined, fixed time. Here we show further data that illustrate why this must be done to ensure accurate mutation measurements. Such accuracy was necessary for our published determination that mismatch repair proteins are not limiting during growth-dependent mutation, but become so during stationary-phase mutation. We review the evidence supporting the idea that stationary-phase reversion of a lac frameshift mutation occurs in an environment of decreased mismatch repair capacity. Those data are substantial. The data presented in the Commentary, in apparent contradiction to this idea, do not justify the conclusion presented there.

Mismatch repair protein MutL becomes limiting during stationary-phase mutation.

Postsynthesis mismatch repair is an important contributor to mutation avoidance and genomic stability in bacteria, yeast, and humans. Regulation of its activity would allow organisms to regulate their ability to evolve. That mismatch repair might be down-regulated in stationary-phase Escherichia coli was suggested by the sequence spectrum of some stationary-phase ("adaptive") mutations and by the observations that MutS and MutH levels decline during stationary phase. We report that overproduction of MutL inhibits mutation in stationary phase but not during growth. MutS overproduction has no such effect, and MutL overproduction does not prevent stationary-phase decline of either MutS or MutH. These results imply that MutS and MutH decline to levels appropriate for the decreased DNA synthesis in stationary phase, whereas functional MutL is limiting for mismatch repair specifically during stationary phase. Modulation of mutation rate and genetic stability in response to environmental or developmental cues, such as stationary phase and stress, could be important in evolution, development, microbial pathogenicity, and the origins of cancer.

Recombination-dependent mutation in non-dividing cells.

Over the past 6 years an unexpected way of making mutations in bacteria has challenged concepts of the genetic mechanisms behind evolution. Mechanistic studies of these so called 'adaptive' mutations are revealing a novel molecular mechanism involving DNA double-strand breaks, genetic recombination, probable DNA polymerase errors, and the possible suspension of mismatch repair during the reversion of a lac frameshift mutation in Escherichia coli. The molecular details of this process are altering our understanding of how mutations form in non-dividing cells.

Adaptive mutation sequences reproduced by mismatch repair deficiency.

Adaptive reversions of a lac frameshift mutation in Escherichia coli are -1 deletions in small mononucleotide repeats, whereas growth-dependent reversions are heterogeneous. The adaptive mutations resemble instability of simple repeats, which, in hereditary colon cancer, in yeast, and in E. coli occurs in the absence of mismatch repair. The postulate that mismatch repair is disabled transiently during adaptive mutation in E. coli is supported here by the demonstration that the growth-dependent mutation spectrum can be made indistinguishable from adaptive mutations by disallowing mismatch repair during growth. Physiologically induced mismatch repair deficiency could be an important mutagenic mechanism in cancers and in evolution.

Adaptive mutation by deletions in small mononucleotide repeats.

Adaptive reversion of a +1 frameshift mutation in Escherichia coli, which requires homologous recombination functions, is shown here to occur by -1 deletions in regions of small mononucleotide repeats. This pattern makes improbable recombinational mechanisms for adaptive mutation in which blocks of sequences are transferred into the mutating gene, and it supports mechanisms that use DNA polymerase errors. The pattern appears similar to that of mutations found in yeast cells and in hereditary colon cancer cells that are deficient in mismatch repair. These results suggest a recombinational mechanism for adaptive mutation that functions through polymerase errors that persist as a result of a deficiency in post-synthesis mismatch repair.

Recombination in adaptive mutation.

The genetic requirements for adaptive mutation in Escherichia coli parallel those for homologous recombination in the RecBCD pathway. Recombination-deficient recA and recB null mutant strains are deficient in adaptive reversion. A hyper-recombinagenic recD strain is hypermutable, and its hypermutation depends on functional recA and recB genes. Genes of subsidiary recombination systems are not required. These results indicate that the molecular mechanism by which adaptive mutation occurs includes recombination. No such association is seen for spontaneous mutation in growing cells.