Improving the thermal stability of lactate oxidase by directed evolution

Lactate oxidase is used in biosensors to measure the concentration of lactate in the blood and other body fluids. Increasing the thermostability of lactate oxidase can significantly prolong the lifetime of these biosensors. We have previously obtained a variant of lactate oxidase from Aerococcus viridans with two mutations (E160G/V198I) that is significantly more thermostable than the wild-type enzyme. Here we have attempted to further improve the thermostability of E160G/V198I lactate oxidase using directed evolution. We made a mutant lactate oxidase gene library by applying error-prone PCR and DNA shuffling, and screened for thermostable mutant lactate oxidase using a plate-based assay. After three rounds of screening we obtained a thermostable mutant lactate oxidase, which has six mutations (E160G/V198I/G36S/T103S/A232S/F277Y). The half-life of this lactate oxidase at 70 °C was about 2 times that of E160G/V198I and about 36 times that of the wild-type enzyme. The amino acid mutation process suggests that the combined neutral mutations are important in protein evolution. Received 15 September 2006; received after revision 21 October 2006; accepted 2 November 2006     

Directed evolution as a tool for understanding and optimizing nucleic acid polymerase function

Polynucleotide polymerases play a crucial role in transmitting genetic information from generation to generation, and they are the most important reagents in biotechnology. Although classical crystal structure analyses as well as biochemical studies have significantly contributed to our understanding of how DNA polymerases function, surprising new insights regarding the importance of certain residues and protein motifs, or of their mutability have been achieved in recent years by evolutionary approaches. Directed evolution has also facilitated the generation of polymerases with tailored substrate repertoires or with stabilities and activities beyond those of their naturally evolved counterparts. Recent new insights in polymerase structure-function relationships and new achievements in the development of tailored polymerases for current methods of nucleic acid synthesis will be summarized in this article. Received 22 April 2005; received after revision 20 July 2005; accepted 27 July 2005     

Kant and the scope of analogy in the life sciences

In the present paper I investigate the role that analogy plays in eighteenth-century biology and in Kant's philosophy of biology. I will argue that according to Kant, biology, as it was practiced in the eighteenth century, is fundamentally based on analogical reflection. However, precisely because biology is based on analogical reflection, biology cannot be a proper science. I provide two arguments for this interpretation. First, I argue that although analogical reflection is, according to Kant, necessary to comprehend the nature of organisms, it is also necessarily insufficient to fully comprehend the nature of organisms. The upshot of this argument is that for Kant our understanding of organisms is necessarily limited. Second, I argue that Kant did not take biology to be a proper science because biology was based on analogical arguments. I show that Kant stemmed from a philosophical tradition that did not assign analogical arguments an important justificatory role in natural science. Analogy, according to this conception, does not provide us with apodictically certain cognition. Hence, sciences based on analogical arguments cannot constitute proper sciences.     

Kant on anatomy and the status of the life sciences

This paper contributes to recent interest in Kant's engagement with the life sciences by focusing on one corner of those sciences that has received comparatively little attention: physical and comparative anatomy. By attending to remarks spread across Kant's writings, we gain some insight into Kant's understanding of the disciplinary limitations but also the methodological sophistication of the study of anatomy and physiology. Insofar as Kant highlights anatomy as a paradigmatic science guided by the principle of teleology in the Critique of the Power of Judgment, a more careful study of Kant's discussions of anatomy promises to illuminate some of the obscurities of that text and of his understanding of the life sciences more generally. In the end, it is argued, Kant's ambivalence with regard to anatomy gives way to a pessimistic conclusion about the possibility that anatomy, natural history, and, by extension, the life sciences more generally might one day become true natural sciences.     

Systematics and evolution of holarctic Pierinae (Lepidoptera). An enzyme electrophoretic approach

Summary Enzyme electrophoretic data show a remarkably high degree of genetic similarity within the European group ofnapi s.l. whereas genetic differences exist at several loci between the European and the North American taxa ofnapi s.l. It is concluded that the European taxa did not differentiate to the species level and form a phylogenetically young group. The North American taxa included in this study are specifically distinct from Europeannapi and separated much earlier. Within these North American taxamarginalis, oleracea andvirginiensis did undergo speciation. The data show a splitting of the genusPieris into three species groups, each genetically differentiated to the same level. The splitting ofPieris into two genera, as suggested by earlier investigators, is not supported here.     

Analogical reflection as a source for the science of life: Kant and the possibility of the biological sciences

In contrast to the previously widespread view that Kant's work was largely in dialogue with the physical sciences, recent scholarship has highlighted Kant's interest in and contributions to the life sciences. Scholars are now investigating the extent to which Kant appealed to and incorporated insights from the life sciences and considering the ways he may have contributed to a new conception of living beings. The scholarship remains, however, divided in its interest: historians of science are concerned with the content of Kant's claims, and the ways in which they may or may not have contributed to the emerging science of life, while historians of philosophy focus on the systematic justifications for Kant's claims, e.g., the methodological and theoretical underpinnings of Kant's statement that living beings are mechanically inexplicable. My aim in this paper is to bring together these two strands of scholarship into dialogue by showing how Kant's methodological concerns (specifically, his notion of reflective judgment) contributed to his conception of living beings and to the ontological concern with life as a distinctive object of study. I argue that although Kant's explicit statement was that biology could not be a science, his implicit and more fundamental claim was that the study of living beings necessitates a distinctive mode of thought, a mode that is essentially analogical. I consider the implications of this view, and argue that it is by developing a new methodology for grasping organized beings that Kant makes his most important contribution to the new science of life.     

Kant and the nature of matter: Mechanics,chemistry, and the life sciences

Kant believed that the ultimate processes that regulate the behavior of material bodies can be characterized exclusively in terms of mechanics. In 1790, turning his attention to the life sciences, he raised a potential problem for his mechanically-based account, namely that many of the operations described in the life sciences seemed to operate teleologically. He argued that the life sciences do indeed require us to think in teleological terms, but that this is a fact about us, not about the processes themselves. Nevertheless, even were we to concede his account of the life sciences, this would not secure the credentials of mechanics as a general theory of matter. Hardly any material properties studied in the second half of the eighteenth century were, or could have been, conceived in mechanical terms. Kant's concern with teleology is tangential to the problems facing a general matter theory grounded in mechanics, for the most pressing issues have nothing to do with teleology. They derive rather from a lack of any connection between mechanical forces and material properties. This is evident in chemistry, which Kant dismisses as being unscientific on the grounds that it cannot be formulated in mechanical terms.     

Oxygen consumption and the evolution of order: negentropy criteria applied to the evolution of ants

Optimization of energy use by evolving organisms, predicted by theoretical extensions of the neo-Darwinian theory, i. contrasted with that of irreversible thermodynamics, which predicts an increase in orderliness and thus an increase in energy consumption per unit of biomass. We compared this index with estimates of social complexity among ant genera and species. Our results show that simple optimization models cannot explain experimental data, and that social complexity correlates differently with negentropy at different levels of analysis. Comparing the genera among Formicidae, workers (not colonies) from genera with highly social species are less negentropic than those of socially primitive ones. At the sub-generic level, social complexity correlated positively with negentropy among species, for major workers inAcromyrmex and for minor workers inAtta. The results illustrate the complexity of thermodynamic criteria in the study of evolution but also hint at their usefulness. In this case, they show that two different evolutionary routes to the complex Attini ant societies may exist.     

Conserved eukaryotic transposable elements and the evolution of gene regulation

Multiple remnants of transposable elements preserved in cis-regulatory modules may represent a record of mutations that were critical to the evolution of gene regulation and speciation. Received 13 August 2007; received after revision 8 October 2007; accepted 23 October 2007     

Effects of chronic caffeine feeding on the activities of oxygen free radical defense enzymes in the growing rat heart and liver

The purpose of the present study was to determine the relationship between concentration of Zn, Cu and Fe, and the catalase, glutathione peroxidase and superoxide dismutase activities in the heart and liver of newborn rats whose dams were fed a diet supplemented with caffeine. Heart Zn levels of the 22- and 30-day-old rats of the caffeine group showed a decrease, whereas liver Zn levels showed an increase compared to the control. Cu levels in the liver at day 22 in the caffeine group were less than in the control. Cu- and Zn-containing superoxide dismutase activities showed an increase in the hearts of the caffeine group compared to the control. The activity of catalase and glutathion peroxidase showed no difference in the heart and liver between the groups. The present study suggests the possible involvement of superoxide dismutase enzyme in the impairment of heart formation as a result of chronic caffeine intake in the early growing period.     

Structure and evolution of the genetic code viewed from the perspective of the experimentally expanded amino acid repertoire in vivo

Much effort has been devoted recently to expanding the amino acid repertoire in protein biosynthesis in vivo. From such experimental work it has emerged that some of the non-canonical amino acids are accepted by the cellular translational machinery while others are not, i.e. we have learned that some determinants must exist and that they can even be anticipated. Here, we propose a conceptual framework by which it should be possible to assess deeper levels of the structure of the genetic code, and based on this experiment to understand its evolution and establishment. First, we propose a standardised repertoire of 20 amino acids as a basic set of conserved building blocks in protein biosynthesis in living cells to be the main criteria for genetic code structure and evolutionary considerations. Second, based on such argumentation, we postulate the structure and evolution of the genetic code in the form of three general statements: (i) the nature of the genetic code is deterministic; (ii) the genetic code is conserved and universal; (iii) the genetic code is the oldest known level of complexity in the evolution of living organisms that is accessible to our direct observation and experimental manipulations. Such statements are discussed as our working hypotheses that are experimentally tested by recent findings in the field of expanded amino acid repertoire in vivo. Received 30 June 1999; accepted 9 July 1999     

Quantum jumps,superpositions, and the continuous evolution of quantum states

The apparent dichotomy between quantum jumps on the one hand, and continuous time evolution according to wave equations on the other hand, provided a challenge to Bohr's proposal of quantum jumps in atoms. Furthermore, Schrödinger's time-dependent equation also seemed to require a modification of the explanation for the origin of line spectra due to the apparent possibility of superpositions of energy eigenstates for different energy levels. Indeed, Schrödinger himself proposed a quantum beat mechanism for the generation of discrete line spectra from superpositions of eigenstates with different energies.However, these issues between old quantum theory and Schrödinger's wave mechanics were correctly resolved only after the development and full implementation of photon quantization. The second quantized scattering matrix formalism reconciles quantum jumps with continuous time evolution through the identification of quantum jumps with transitions between different sectors of Fock space. The continuous evolution of quantum states is then recognized as a sum over continually evolving jump amplitudes between different sectors in Fock space.In today's terminology, this suggests that linear combinations of scattering matrix elements are epistemic sums over ontic states. Insights from the resolution of the dichotomy between quantum jumps and continuous time evolution therefore hold important lessons for modern research both on interpretations of quantum mechanics and on the foundations of quantum computing. They demonstrate that discussions of interpretations of quantum theory necessarily need to take into account field quantization. They also demonstrate the limitations of the role of wave equations in quantum theory, and caution us that superpositions of quantum states for the formation of qubits may be more limited than usually expected.     

Ancient origin of reggie (flotillin), reggie-like, and other lipid-raft proteins: convergent evolution of the SPFH domain

Reggies (flotillins) are detergent-resistant microdomains involved in the scaffolding of large heteromeric complexes that signal across the plasma membrane. Based on the presence of an evolutionarily widespread motif, reggies/flotillins have been included within the SPFH (stomatin-prohibitin-flotillin-HflC/K) protein superfamily. To better understand the origin and evolution of reggie/flotillin structure and function, we searched databases for reggie/flotillin and SPFH-like proteins in organisms at the base and beyond the animal kingdom, and used the resulting dataset to compare their structural and functional domains. Our analysis shows that the SPFH grouping has little phylogenetic support, probably due to convergent evolution of its members. We also find that reggie/flotillin homologues are highly conserved among metazoans but are absent in plants, fungi and bacteria, where only proteins with ‘reggie-like’ domains can be found. However, despite their low sequence similarities, reggie/flotillin and ‘reggie-like’ domains appear to subserve related functions, suggesting that their basic biological role was acquired independently during evolution. Received 21 September 2005; received after revision 14 November 2005; accepted 21 November 2005     

高速铁路基础结构动态性能演变及服役安全的基础科学问题

中国高速铁路已经由大规模建造进入长期安全稳定运营阶段,如何科学维护庞大规模的高速铁路运营线路,使高速铁路长期保持高安全、高稳定、高可靠的运营品质,是我国现阶段面临的重大课题.在分析总结中国高速铁路基础结构构造特点及其复杂运营环境特点的基础上,提出了高速铁路基础结构动态性能演变及服役安全的三个基础科学问题:环境与动载耦合重复作用下高速铁路基础结构关键工程材料与结构动态性能演变,高速铁路基础结构累积变形与轨面几何形态的映射关系,基础结构劣化与高速列车-基础结构耦合系统动态性能的相互影响及演化机制;回顾评述了国内外在此方面的研究现状与最新进展;指出了本领域今后的发展趋势以及需要重点关注和加强的基础研究工作.     

Detection of caprine arthritis-encephalitis- and maedi-visna viruses using the polymerase chain reaction

Summary The polymerase chain reaction (PCR) was used to demonstrate proviral DNA of lentiviruses of small ruminants in cultured cells. Primers for the Taq polymerase were selected in the GAG gene of Icelandic maedi-visna virus and POL gene of caprine arthritis-encephalitis (CAE) virus. Using PCR, proviral DNA of CAE virus was detected at 1 day post infection, 4 days beforeviral protein could be demonstrated using a sensitive immunoblotting protocol and 6 days before the appearance of syncytia. Primers derived from the published sequence of CAE virus successfully primed for the synthesis of homologous virus and Icelandic maedi-visna viruss but not for maedi-visna virus isolated in The Netherlands. In contrast, primers derived from the GAG region of Icelandic maedi-visna virus allowed the amplification of DNA of homologous virus, maedi-visna virus isolated in The Netherlands as well as CAE virus.     

Implications of toxins in the ecology and evolution of plant pathogenic microorganisms: Bacteria

This review attempts to rationalise what is known about bacterial phytotoxins and associate it with the ecology and possible evolution of the producing organisms. Study of non-toxin producing variants gives insight into the ecological role of the toxin. Elucidation of chemical structures of phytotoxins has shown that many exist as families of analogous compounds. Studies on the variation of chemical structures and how they are distributed across species and genera can lead to development of hypotheses on evolutionary relationships. Knowledge on biosynthetic pathways to tosins allows recognition of specific enzymatic steps involved in developing the characteristic features of the structures. Phytotoxins often have a potent biochemical activity, and in some cases the producing organism has associated mechanisms to prevent action of the toxin upon itself; in such cases toxigenesis is clearly not a chance event. The various aspects of bacterial toxigenesis indicate that bacterial phytotoxins are special secondary metabolic products that play beneficial roles to the producing organisms in their various ecological niches.     

Sea urchin embryo as a model for analysis of the signaling pathways linking DNA damage checkpoint,DNA repair and apoptosis

DNA integrity checkpoint control was studied in the sea urchin early embryo. Treatment of the embryos with genotoxic agents such as methyl methanesulfonate (MMS) or bleomycin induced the activation of a cell cycle checkpoint as evidenced by the occurrence of a delay or an arrest in the division of the embryos and an inhibition of CDK1/cyclin B activating dephosphorylation. The genotoxic treatment was shown to induce DNA damage that depended on the genotoxic concentration and was correlated with the observed cell cycle delay. At low genotoxic concentrations, embryos were able to repair the DNA damage and recover from checkpoint arrest, whereas at high doses they underwent morphological and biochemical changes characteristic of apoptosis. Finally, extracts prepared from embryos were found to be capable of supporting DNA repair in vitro upon incubation with oligonucleotides mimicking damage. Taken together, our results demonstrate that sea urchin early embryos contain fully functional and activatable DNA damage checkpoints. Sea urchin embryos are discussed as a promising model to study the signaling pathways of cell cycle checkpoint, DNA repair and apoptosis, which upon deregulation play a significant role in the origin of cancer. Received 10 April 2007; accepted 23 April 2007     

Elucidation of the relationship between enzyme activity and internal motion using a lysozyme stabilized by cavity-filling mutations

We investigated the activity and the internal motions of a stabilized mutant hen lysozyme (HEL) in which the residues M12 and L56 were mutated to L and F, respectively (LF mutant HEL). The result of the activity measurements against glycol chitin at various temperatures suggested that the temperature dependence of the activity of LF mutant HEL shifted to the high-temperature side compared with that of wild-type HEL. The detailed internal motions of LF mutant HEL in the absence and presence of a substrate analogue, (NAG)₃, were examined by model-free analysis at 35°C. The results showed that the internal motions of LF mutant HEL in the presence of (NAG)₃ were drastically restricted compared with those in wild-type HEL. Our findings thus suggested that the mutation to the stabilized lysozyme restricted internal motions required for the enzymatic reaction.Received 8 February 2005; accepted 10 March 2005Y. Yoshida and T. Ohkuri contributed equally to this work.